Process for producing farnesylated dibenzodiazepinone by fermentation

ABSTRACT

The present invention provides a scalable process for producing a concentrate containing a mass of a farnesylated dibenzodiazepinone by fermenting in an aqueous culture medium a strain of a microorganism that is capable of producing the farnesylated dibenzodiazepinone, upon completion of fermentation harvesting the fermentation broth and extracting the fermentation broth to provide an extract, and thereafter treating the extract to form the concentrate. The concentrate so produced may be utilized in downstream processes for producing pharmaceutical compounds. A strain of a  Micromonospora  species capable of producing a farnesylated dibenzodiazepinone at a high yield rate is provided, together with culture media for culturing microorganisms, and fermentation conditions for production of the farnesylated dibenzodiazepinone of the concentrate.

The present application claims benefit under 35 U.S.C. §119(e) of U.S.provisional patent application No. 60/791,903, filed Apr. 14, 2006, theentire disclosure of which is herein incorporated by reference.

FIELD OF INVENTION

The present invention relates to a process for preparing a concentratecomprising a mass of a farnesylated dibenzodiazepinone, namely ECO-4601,from a fermentation broth. The invention further relates to aconcentrate comprising a mass of a farnesylated dibenzodiazepinone,namely ECO-4601, wherein said concentrate may be processed for theprovision of a medicament for administration to a mammal in need of suchmedicament for the treatment of a disease condition such as cancer.

BACKGROUND

The compound ECO-04601 (hereinafter ECO-4601) is a novel farnesylateddibenzodiazepinone, namely10-farnesyl-4,6,8-trihydroxy-dibenzodiazepin-11-one, having thestructure of Formula I as shown below.

ECO-4601 has been isolated from fermentation cultures of a novel strainof the actinomycete Micromonospora sp. as disclosed in U.S. patentapplication Ser. No. 10/762,107 filed Jan. 21, 2004 (published Feb. 24,2005 as US 2005-0043297 A1, issued as U.S. Pat. No. 7,101,872 on Sep. 5,2006), also published Aug. 5, 2004 as PCT International ApplicationWO2004/065591. The structure of ECO-4601 has been subsequently disclosedby Charan et al. (2004), J. Nat. Prod., vol. 67, pages 1431-1433, thecompound having been extracted from solids collected from a fermentationbroth of Micromonospora strain DPJ12 that was isolated from the marineascidian Didemnum proliferum Kott. at Shishijima Island, Japan. Charanet al. have referred to the isolated farnesylated dibenzodiazepinone as“diazepinomicin”. The compound ECO-4601 has also been subsequentlydisclosed by Igarashi et al. (2005), J. Antibiot., vol. 58, pages350-352, this group having isolated the compound by extraction of afermentation broth of a culture of an actinomycete, Micromonospora sp.TP-A086, that was isolated from a soil sample collected in Osawano,Japan. ECO-4601 has been shown to possess anti-bacterial and anti-canceractivities, and results from animal model studies indicate an in vivoanti-cancer potency of ECO-4601 (see U.S. patent application Ser. No.10/951,436 filed Sep. 27, 2004 (published May 19, 2005 as US 2005-010736A1, issued as U.S. Pat. No. 7,186,713 on Mar. 6, 2007), and U.S. patentapplication Ser. No. 11/130,295 filed May 16, 2005 (published Apr. 13,2006 as US 2006-0079508 A1)).

Production of limited amounts of ECO-4601 via fermentation methods havebeen disclosed. U.S. patent application Ser. No. 11/130,295 disclosesthat ECO-4601 can be produced by inoculating a culture medium with asample of a microorganism capable of producing ECO-4601, such asMicromonospora strain 046-ECO11 or strain [S01]046, and thereafterincubating the inoculated culture with aeration by agitation such as byshaking on a water bath or in a fermentor, or by injection of air,oxygen or an appropriate gaseous mixture into the culture medium.Incubation can last for a period of about 3 to 4 days at temperatures ofabout 18 to about 40° C., and at a pH of about 6 to about 9, in asuitable medium containing assimable sources of carbon, nitrogen,optional inorganic salts and other known growth factors. Upon completingcultivation, ECO-4601 can be isolated from the culture medium bytechniques known in the art such as centrifugation, adsorption,chromatography, and filtration. Organic solvents such as ethyl acetate,n-butanol, n-butyl acetate or 4-methyl-2-pentanone may be mixed with thecultivated culture medium and the organic layer separated bycentrifugation followed by solvent removal by evaporation to dryness, orby evaporation to dryness under a vacuum, to yield an ECO-4601containing residue. Downstream purification can be performed byoptionally reconstituting the residue with ethanol, ethyl acetate,methanol or a mixture thereof, and re-extraction in a two-phase systemusing a suitable organic solvent such as hexane, acetonitrile, ethylacetate, methanol and carbon tetrachloride, methylene chloride or amixture thereof, followed by further purification by techniques known inthe art, such as chromatography.

Fermentation methods for producing limited amounts of the farnesylateddibenzodiazepinone of the present invention have been described in theart. For example, Igarashi et al. (supra) described a process wherein a5 liter fermentation broth was extracted with 1-butanol and the organiclayer concentrated in vacuo to yield an oily extract of 17 grams.Chromatographic purification (silica gel followed by LH-20) of theextract yielded 16 mg of light yellow needles. In Charan et al. (supra),a process was described wherein a 4 liter fermentation broth ofMicromonospora DPJ12 was centrifuged and the resulting cell mass andHP20 resin (50 g/L of broth) were washed with water and extracted withmethanol (3×250 mL). The combined methanol extracts were concentrated invacuo and extracted with ethyl acetate (4×60 mL) to yield an extract of278 milligrams. Further purification (Sephadex LH-20 columnchromatography followed by reversed-phase HPLC) yielded 4.8 mg of thediazepinomicin product.

For development of ECO-4601 into a commercial pharmaceutical product,fermentation production of the compound will be required on a scale(s)to generate quantities of the compound to meet sufficiently supplyrequirements for clinical trial testing regimens, and thereafter forsubsequent commercial manufacturing of formulations and dosages thatwill be prescribed to patients in need of treatment. As such, thereremains the need for a robust and commercially-practical fermentationprocess pertaining to the fermentation of Micromonospora sp. for theproduction of quantities of ECO-4601 useful for clinical trials andcommercial production of pharmaceutical products. Advantageously, thefermentation process should provide several benefits in terms of beingscalable from a laboratory level to various levels commercialproduction, while concomitantly not requiring an increased number offermentation vessels or other pieces of equipment and/or steps involvedin downstream processing and purification steps, nor require increasedvolumes of an extraction solvent or solvents beyond that directlyproportional to an increased fermentation volume. As well, there existsa need for a fermentation process for the production of ECO-4601 thatprovides for a high yield rate of the compound from a fermentationbroth, and which allows for the compound to be efficiently extractedfrom the fermentation broth and provided in a form, preferably aconcentrated form, that is stable, easily transportable, and which canthereafter be easily subjected to downstream processing for productionof crystalline forms of ECO-4601 that may thereafter be formulated intopharmaceutically-acceptable compositions for use in patients requiringneed of such compositions.

SUMMARY OF THE INVENTION

The present invention provides a process for recovering andconcentrating a farnesylated dibenzodiazepinone of structural Formula I

comprising the steps of: a) fermenting a strain of a microorganismcapable of producing said farnesylated dibenzodiazepinone in an aqueousculture medium thereby producing a fermentation broth comprising saidfarnesylated dibenzodiazepinone of structural Formula I; b) adjustingsaid fermentation broth to allow said farnesylated dibenzodiazepinone toassociate with a particulate matter present in said broth; c) harvestingsaid particulate matter from said broth for obtaining a harvestedparticulate matter; d) extracting said harvested particulate matter witha volume of a suitable organic solvent in a proportion of about 2:1 toabout 5:1 to said harvested particulate matter to thereby form anextract; and e) treating said extract to form a first concentratecomprising said farnesylated dibenzodiazepinone, wherein said firstconcentrate comprises said farnesylated dibenzodiazepinone at aconcentration greater than about 50-fold than said farnesylateddibenzodiazepinone in said fermentation broth of a), and wherein saidfarnesylated dibenzodiazepinone is recovered from said fermentationbroth of a) in an amount of at least about 50% of the amount of saidfarnesylated dibenzodiazepinone in said fermentation broth. In yetanother aspect, the process of the present invention further comprisesprocessing said first concentrate in order to reduce a level of animpurity in said first concentrate to thereby produce a secondconcentrate. In one aspect of the present invention, the secondconcentrate is substantially free of molecules other than thefarnesylated dibenzodiazepinone of Formula I. In yet a still furtheraspect, the process of the present invention even further comprises astep of crystallizing from said second concentrate the farnesylateddibenzodiazepinone of Formula I to thereby produce a crystallinefarnesylated dibenzodiazepinone of Formula I suitable for use in thepreparation of a pharmaceutical formulation.

In yet a further aspect of the present invention, the farnesylateddibenzodiazepinone is recovered from said fermentation broth of a) in anamount of at least about 60% of the amount of said farnesylateddibenzodiazepinone in said fermentation broth, and in yet another aspectthe farnesylated dibenzodiazepinone is recovered from said fermentationbroth of a) in an amount of at least about 65% of the amount of saidfarnesylated dibenzodiazepinone in said fermentation broth.

In yet another aspect of the process of the present invention, theparticulate matter comprises the fermented microorganism present in thefermentation broth. In still yet a further aspect of the process of thepresent invention, the particulate matter further comprises an adsorbentresin. In yet another aspect of the process of the present invention,the fermentation broth is adjusted to a pH value of about 2 to about 4,and in yet another aspect of the process of the present invention, thefermentation broth is further adjusted to a temperature range of about2° C. to about 10° C., while in yet another aspect of the process of thepresent invention, the fermentation broth is maintained at thetemperature range for a period of about 16 hours to about 72 hours. Instill yet another aspect of the process of the present invention, thefermentation is completed in from about 48 hours to about 110 hours.

In a still further aspect of the process of the present invention, themicroorganism is a bacterium, and still further, the bacterium is anActinomycete, and yet further the Actinomycete is a Micromonospora,Streptomyces or a Rhodococcus species, and yet further the Actinomyceteis Micromonospora sp. [S01]046 having IDAC accession number 231203-01 orMicromonospora sp. [S01U02]046 having IDAC accession number 070905-01.

In a further aspect of the process of the present invention, thesuitable organic solvent comprises at least one lower alkyl alcohol, andin a further aspect the lower alkyl alcohol is methanol, or ethanol, orpropanol, or iso-propanol or butanol or a mixture of two of these loweralkyl alcohols. In yet a further aspect, the suitable organic solvent isethyl acetate or acetonitrile.

In another aspect of the process of the present invention, the aqueousculture medium in which the strain of the microorganism is fermented isAP, CA, DZ, GP, HI, JA, MI, PI, QI, QP or YB, and in yet another aspectthe aqueous culture medium is QB, MA, KH, RM, JA, FA, or HI.

In another aspect of the process of the present invention, theharvesting of the particulate matter from the broth is performed usingan ultrafiltration system, and in yet another aspect, theultrafiltration system has one or more filter elements having a poresize of about 0.2 to about 0.45 micrometers. In a still further aspectof the process of the present invention, the harvesting of theparticulate matter from the broth is performed by centrifugation.

In another aspect of the process of the present invention, theextraction of the harvested particulate matter comprises one to threerounds of extraction using the suitable organic solvent, and in yet afurther aspect, the extraction further comprises circulating theparticulate matter together with the suitable organic solvent in asystem at a flow rate of about 30 Hz to about 50 Hz, and at atemperature of about 39° C. to about 45° C., and yet still further, thecirculation is for a period of about 50 minutes to about 120 minutes. Inyet a further aspect, the extraction comprises subjecting theparticulate matter together with the suitable organic solvent to acentrifugation treatment. In yet a still further aspect of the processof the present invention, the volume of the suitable organic solventutilized for the extraction is calculated in proportion to the harvestedparticulate matter mass (volume:mass) or in proportion to the harvestedparticulate matter volume (volume:volume).

In another aspect of the process of the present invention, the treatingof the extracted, harvested particulate matter is an evaporationtreatment, and in yet a further aspect, the evaporation treatment isconducted under a reduced pressure, and in yet a further aspect, theevaporation is performed while incubating the extract with an adsorbentresin, for example, HP20 resin and wherein evaporation treatment isconducted under a reduced pressure.

In another aspect of the process of the present invention, the treatingof the extracted, harvested particulate matter comprises incubating theextract with an absorbent resin to form a mixture, followed by anaddition of water to the mixture to displace the farnesylateddibenzodiazepinone of Formula I onto the resin. In yet a further aspect,the adsorbent resin mixed with the extract is provided in a ratio (W/W)of about 10 times to about 40 times to the farnesylateddibenzodiazepinone of Formula I in the extract. In yet another aspect ofthe process of the present invention, the water is added to the mixtureat a volumetric rate (V/V) of about 0.5% to about 20% of the volume ofthe mixture, and in yet another aspect, volume flow rate is about 0.5%to about 5% of the volume of the mixture. In a still further aspect, thewater added to the mixture is of a total volume of about 1.0 to about1.5 times the volume of the mixture, and in yet a further aspect, thetotal volume of water added to the mixture is of about 1.2 to about 1.5times the volume of the mixture.

In a further embodiment, the present invention provides for aconcentrate comprising the farnesylated dibenzodiazepinone of structuralFormula I

wherein said concentrate is obtained according to the method of thepresent invention.

In another aspect, the concentrate that is provided by the presentinvention comprises the farnesylated dibenzodiazepinone of Formula I,wherein the farnesylated dibenzodiazepinone of Formula I is recoveredfrom said fermentation broth of a) in an amount of at least about 60% ofthe amount of said farnesylated dibenzodiazepinone in said fermentationbroth, and in yet another aspect the farnesylated dibenzodiazepinone ofFormula I is recovered from said fermentation broth of a) in an amountof at least about 65% of the amount of said farnesylateddibenzodiazepinone in said fermentation broth.

In a further embodiment, the present invention provides for amicroorganism capable of producing, during a fermentation period in avolume of an aqueous culture medium, a farnesylated dibenzodiazepinoneof structural Formula I

at a yield rate of about 0.073 mg/L/hour to about 5.06 mg/L/hour asaveraged over the fermentation period. In a further aspect of thepresent invention, the yield rate of the microorganism is of about 2.15mg/Uh to about 5.06 mg/Uh as averaged over the fermentation period, andin yet a still further aspect of the present invention, the yield rateof the microorganism is of about 3.00 mg/Uh to about 5.06 mg/L/h asaveraged over the fermentation period. In a further aspect, themicroorganism is a bacterium, and even more particularly, the bacteriumis an Actinomycete, and even still more particularly, the Actinomyceteis a Micromonospora, Streptomyces or a Rhodococcus species, and stillmore particularly, the Actinomycete is Micromonospora sp. [S01]046having IDAC accession number 231203-01 or Micromonospora sp. [S01U02]046having IDAC accession number 070905-01.

In yet a further embodiment, the invention provides for an Actinomycetespecies, namely, Micromonospora sp. [S01U02]046, having IDAC accessionnumber 070905-01.

In a further embodiment, the present invention provides for variousculture medium compositions for culturing bacteria, and methods forculturing bacteria, and in which in yet another aspect of the presentinvention such media and methods may be utilized for growing bacteriaunder aqueous conditions, for example, so as to produce a fermentationbroth culture. In one aspect, the culture medium composition comprises10 g/L glucose, 20 g/L soluble starch, 5 g/L yeast extract, 5 g/LBacto-peptone and 2 g/L CaCO₃. In another aspect, the culture mediumcomposition comprises 10 g/L glucose, 40 g/L potato dextrin, 5 g/L yeastextract, 5 g/L Bacto-peptone and 2 g/L CaCO₃. In another aspect, theculture medium composition comprises 10 g/L glucose, 25 g/L solublestarch, 5 g/L yeast extract, 5 g/L Bacto-peptone and 3 g/L CaCO₃. In yetstill another aspect, the culture medium comprises 12 g/L glucose, 10g/L potato dextrin, 5 g/L corn steep liquor, 10 g/L Pharmamedia and 4g/L Proflo oil. In yet a still further aspect, the culture mediumcomprises 20 g/L potato dextrin, 8.34 g/L yeast extract, 30 g/Lglycerol, 2.5 g/L Bacto-peptone and 3 g/L CaCO₃.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 to 4: show the different steps involved in the biosyntheticpathway of ECO-04601. Each of FIGS. 1 to 4 shows the three biosyntheticloci A, B and C where ORFs are represented by arrows. Highlighted ORFsare involved in the steps described in the schematic diagram. Thebiosynthetic enzymes involved in the steps depicted in schematicdiagrams are indicated by their family designation and the respectiveORF number in each of Loci A, B and C (e.g., 8/7/7).

FIG. 1: shows a schematic diagram of the biosynthetic pathway for theproduction of farnesyl-diphosphate, providing the farnesyl group ofECO-04601.

FIG. 2: shows a schematic diagram of the biosynthetic pathway for theproduction of 3-hydroxy-anthranilate-adenylate precursor of thedibenzodiazepinone group.

FIG. 3: shows a schematic diagram of the biosynthetic pathway for theproduction of 2-amino-6-hydroxy-[1,4]benzoquinone precursor of the coredibenzodiazepinone.

FIG. 4: shows a schematic diagram of the biosynthetic pathway for theassembly of the ECO-04601 precursors, farnesyl-diphosphate,3-hydroxy-anthranilate-adenylate and2-amino-6-hydroxy-[1,4]benzoquinone.

FIG. 5 is a flowchart depicting a plurality of steps involved in aprocedure for the preparation of master and working seed stocks for usein a pilot fermentation production;

FIG. 6 is a flowchart depicting a plurality of steps involved in aprocedure for the preparation production of seed flask fermentations,inoculum fermentations for use with pilot plant fermentations, and apilot plant fermentation;

FIG. 7 is a flowchart depicting a plurality of steps for adjustment of afermentation broth prior to harvesting, harvesting of particulatematter, extracting harvested particulate matter and treating an extractto form a concentrate.

FIG. 8 is a flowchart depicting a plurality of steps involved in aprocedure for the displacement of ECO-4601 from pooled extracts onto aresin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a scalable process for the fermentationproduction of a farnesylated dibenzodiazepinone

I. Definitions

Unless otherwise defined all technical and scientific terms used hereinhave the meaning as commonly understood by a person skilled in the artto which this invention belongs.

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims are provided below.

As used herein, the term “farnesylated dibenzodiazepinone” refers to aclass of dibenzodiazepinone compounds containing a farnesyl moiety. Theterm includes, but is not limited to, the exemplified compound of thepresent invention, 10-farnesyl-4,6,8-trihydroxy-dibenzodiazepin-11-one,which is referred to herein as “ECO-04601”. As used herein, the term“farnesylated dibenzodiazepinone” includes compounds of this class thatcan be used as intermediates in chemical syntheses.

As used herein, the term “concentrate” refers to a liquid, a semi-solidor a solid mass comprising, in part, a mass of a farnesylateddibenzodiazepinone, namely ECO-4601. If in the form of a semi-solid orsolid mass, the concentrate may be substantially free of water and/orsubstantially free of any solvent or solvents utilized to produce theconcentrate. It is to be understood that the concentrate may take a formof a highly viscous paste, whether in a semi-solid or solid form. Aconcentrate in a solid form that is substantially free of any liquid orliquids may be provided mixed with an adsorbent resin.

As used herein the term “first concentrate” refers to a concentratecomprising, in part, the farnesylated dibenzodiazepinone of Formula I,namely ECO-4601, wherein said first concentrate is formed by treating anextract of a fermentation broth comprising the farnesylateddibenzodiazepinone of Formula I. The first concentrate, or a combinationof such first concentrates, may thereafter be subjected to processingfor reducing a level of an impurity present in the first concentrate,for example by being processed in a column cleanup procedure comprisingat least one round of cleanup, and so as to result in the formation of asecond (2^(nd)) concentrate comprising the farnesylateddibenzodiazepinone of Formula I. The second concentrate may besubstantially free of other molecules other than the farnesylateddibenzodiazepinone of Formula I. The second concentrate may thereafterbe subjected to a crystallization process for producing crystallineECO-4601 suitable for use in the preparation of a pharmaceuticalformulation or formulations.

As used herein, the term “ratio (W/W)” refers to a ratio of two massesin relative proportion to each other. Preferably, the term refers to amass of a farnesylated dibenzodiazepinone, namely ECO-4601, present in aconcentrate relative to a mass of ECO-4601 present in a fermentationbroth. Further, the term “ratio (WAN)” includes, and is interchangeablewith, the expression of such a ratio in terms of a percentage of the twomasses relative to each other, for example, by expression of a mass ofECO-4601 present in a concentrate as a percentage of a mass of ECO-4601present in a fermentation broth or combined fermentation broths. Thefermentation broth may be a single broth, or may be a combination of twoor more separate fermentation broths that have been combined prior tobeing extracted. It is to be understood that equivalent mass units willbe employed in describing an amount of ECO-4601 present in theconcentrate relative to the mass of ECO-4601 present in the fermentationbroth, for example, micrograms (μg) to micrograms (μg), milligrams (mg)to milligrams (mg), grams (g) to grams (g), or kilograms (kg) tokilograms (kg). As such, it is to be understood that the mass unitsutilized to express the ratio (W/W) are not restricted to a particularorder or size of units (e.g. only kg), and that all are understood to beincluded when referring to a ratio W/W of weight of ECO-4601 in theconcentrate (a second mass) to weight of ECO-4601 in the fermentationbroth (a first mass).

As used herein, a “strain” means a cell or population of cells that hasthe same general characteristics of a given type of organism, forexample a bacterium, or of a particular genus, species, and serotype. Ifthe strain comprises a population of cells, it will be recognized bythose of skill in the art that the population will be descendant from asingle organism or pure culture isolate.

As used herein, the term “microorganism capable of producing afarnesylated dibenzodiazepinone” refers to a microorganism that carriesgenetic information necessary to produce a farnesyl dibenzodiazepinonecompound, whether or not the organism naturally produces the compound.The terms apply equally to organisms in which the genetic information toproduce the farnesyl dibenzodiazepinone compound is found in theorganism as it exists in its natural environment, and to organisms inwhich the genetic information is introduced by recombinant techniques.Specific organisms contemplated herein include, without limitation,organisms of the family Micromonosporaceae, of which preferred generainclude Micromonospora, Actinoplanes and Dactylosporangium; the familyStreptomycetaceae, of which preferred genera include Streptomyces andKitasatospora; the family Pseudonocardiaceae, of which preferred generaare Amycolatopsis and Saccharopolyspora; the family Actinosynnemataceae,of which preferred genera include Saccharothrix and Actinosynnema; andspecies of the genus Rhodococcus; however the terms are intended toencompass all organisms containing genetic information necessary toproduce a farnesyl dibenzodiazepinone compound. A preferred producer ofa farnesyl dibenzodiazepinone compound includes Micromonospora sp.strain [S01U02]046, a deposit of which was made on Sep. 7, 2005, withthe International Depository Authority of Canada (IDAC), Bureau ofMicrobiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba,Canada R3E,3R2, under Accession No. IDAC 070905-01.

As used herein, an “aqueous medium” means a medium containing one moresources of assimable carbon, nitrogen and inorganic salts and which iscapable of supporting growth of a microorganism capable of synthesizingECO-4601.

As used herein, the term “adjusting” refers to a change induced, uponcompletion of a fermentation culture period, in one or more of aphysical or a chemical condition or conditions of a fermentation brothcomprising a farnesylated dibenzodiazepinone. An adjustment of achemical condition of the fermentation broth includes, but is notlimited to, inducing an acidification of the fermentation broth. Anadjustment of a physical condition of the fermentation broth includes,but is not limited to, a reduction in the temperature of thefermentation broth. Any particular adjustment may be accomplished usingtechniques or reagents known in the art, for example, differentmechanical means for lowering the temperature of a fermentation broth.The term is to equally apply to the adjusting of a condition orconditions that occurs by a series of graded or step-wise adjustmentsversus a single adjustment. As well, the term is to be understood toinclude the contemporaneous adjustment of more than one condition,whether or not the adjustment of each condition occurs by a series ofgraded or step-wise adjustments versus a single adjustment.

As used herein, the term “a particulate matter” refers to minuteparticles of one or more types that are present in the fermentationbroth comprising the farnesylated dibenzodiazepinone. The term includes,but is not limited to, the microorganism that is capable of producingthe farnesylated dibenzodiazepinone, and which by default would bepresent in the fermentation broth. It is to be understood that the termequally applies to an adsorbent resin that would be capable of bindingat least the farnesylated dibenzodiazepinone. The adsorbent resin may bepresent in the fermentation broth upon initiation of the fermentationprocess, or may be added to the fermentation broth upon expiry of anallotted time or upon the fermentation broth reaching a pre-determinedcondition such as, for example, a particular degree of density or age. Anumber of types of adsorbent resins are known in the art and may be usedin the present invention. Such resins include, but are not limited to,XAD 2, XAD 4, XAD 7, XAD 8 and XAD 16 (Rohm and Haas); HP20, HP20S,HP20SS and SP70 (Diaion®); and reverse phase silicas such as C-8, C-10and C-18.

As used herein, the term “associate with” refers to a chemical orphysical interaction that may occur between a molecule of a farnesylateddibenzodiazepinone and a surface or surfaces of a particulate matterpresent in a fermentation broth such that the farnesylateddibenzodiazepinone is retained or substantially retained by theparticulate matter. The term includes, but is not limited to, weakinteractions that may occur between molecules of the farnesylateddibenzodiazepinone and a surface of a particulate matter present in thefermentation broth, for example, hydrogen bonding, electrostaticattractions, van der Waals bonding, hydrophobic attractions, orpartitioning. The term will be understood to include the adsorption ofthe farnesylated dibenzodiazepinone by a synthetic adsorbent resin, suchas for example, Diaion® HP20 resin.

As used herein, the term “suitable organic solvent” refers to an organicsolvent in which the farnesylated dibenzodiazepinone, namely ECO-4601,is readably soluble. By readily soluble, it is meant that the solventwill dissolve a mass of ECO-4601 in sufficient concentration that avolume of the solvent required to extract a slurry provided from theECO-4601-containing fermentation broth of the present invention will bein a range of about twice to about five times of a volume of the slurry.It will be understood that the term equally applies to a mixture oforganic solvents in which the farnesylated dibenzodiazepinone, namelyECO-4601, is readably soluble. A choice of a particular suitable organicsolvent, or a mixture of suitable organic solvents, for extracting thefarnesylated dibenzodiazepinone from the slurry may be dependent upon anumber of factors in combination with the solubility of the farnesyldibenzodiazepinone in the solvent, and such factors may include but arenot limited to a solvent's cost, availability, ease of handling,environmental impact, hazardous potential (e.g. explosivity) anddisposability. As will be appreciated by those of skill in the art, anumber of different suitable organic solvents may be selected.Preferably, the suitable organic solvent would be a one-carbon to afour-carbon solvent, or an aromatic solvent. More preferably, thesuitable organic solvent or mixture of suitable organic solvents wouldbe selected from one or more the solvent families or particularsolvents, as follows: lower alkyl alcohols (e.g., methanol, ethanol,propanol, n-butanol, isopropanol, propylene glycol); acetonitrile;toluene; oxygen-containing organic solvents such as dialkyl ketones(e.g. acetone, 2-butanone), tetrahydrofuran, dioxane, alkyl acetates(e.g. ethyl acetate, iso-propyl acetate, butyl acetate); and dialkylethers (e.g. di-isopropyl ether, tert-butyl methyl ether).

As used herein, the term “volume of a suitable organic solvent in aproportion” refers to a volumetric amount of a solvent as measured inrelation to a mass or a volume of the solute that is to be mixed withthe solvent. It is to be understood that for the purpose of the presentinvention, equivalent volume units will be employed in describing avolume of suitable organic solvent relative to a volume or a mass of aparticulate matter that is harvested from a fermentation broth. Forexample, for a 1 L volume of particulate matter, a 3 L volume of thesuitable organic solvent would be utilized, and if a 100 gram mass ofparticulate material was harvested from the fermentation broth a 300 mLvolume of the suitable organic solvent would be utilized. As will beappreciated by those of skill in the art, the harvested particulatematter may be subjected to two or more rounds of extraction with anequal or non-equal volumes of the suitable organic solvent, solvents, ormixtures thereof, per extraction. Further, it will be understood bythose of skill in the art, that the suitable organic solvent may be amixture of two or more organic solvents, provided that the farnesylateddibenzodiazepinone, namely ECO-4601, is soluble in the mixture.

As used herein, the term “treating” refers to the subjection of anextract of the present invention to a physical or chemical treatment ora combination thereof. The physical or chemical treatment, orcombination thereof, may occur in the form of a physical or chemicalprocess that is applied to the extract, wherein the process in partrequires an incubation of the extract with one or more particularsubstances. The physical or chemical treatment, or combination thereof,should be sufficient to achieve a removal or a substantial removal ofthe suitable organic solvent, solvents, or mixtures thereof from theextract. The term includes, but is not limited to, an evaporationprocess, preferably when said evaporation process is conducted under areduced pressure, and more preferably when said evaporation process isconducted in conjunction with a warming of the extract. Further, theterm includes an addition of an adsorbent resin to the extract, whethersuch addition is an alternative to or in conjunction with the subjectionof the extract to an evaporation process. As well, the term includes amicrofiltration to remove a bulk of the solvent, followed by evaporationto dryness. As well, the term includes a centrifugation to separate, forexample, mycelia from a fermentation broth and also to aid in anextraction of a farnesylated dibenzodiazepinone from mycelia in thepresence of the solvent.

As used herein, the term “displacement” means to move or shift an objector molecule(s), for example molecules of a farnesylateddibenzodiazepinone, from an environment or condition to a differentenvironment, condition, or state, and may include a change in thephysical or chemical state of such a molecule(s), for example, byperformance of a step or series of steps, whether such step or stepsis/are a mechanical or chemical step, so as to induce a removal of suchmolecule(s) from a solution or slurry. Removal of such molecule(s) fromsolution may be achieved, for example, by displacement of suchmolecule(s) from a solution or slurry onto an adsorbent resin.

As used herein, the term “substantially free of other molecules” means aconcentrate or compound or mixture comprising the farnesylateddibenzodiazepinone of Formula I that is at least 60%, at least 70%, atleast about 80%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about99% of 100% pure farnesylated dibenzodiazepinone of Formula I, measuredby weight.

II. Production of a Farnesylated Dibenzodiazepinone by Fermentation

A. Microorganisms, Genes and Recombinant Microorganisms

i) Microorganisms

The farnesyl dibenzodiazepinone compounds of the present invention maybe biosynthesized by various microorganisms. Microorganisms that maysynthesize the compounds of the present invention include but are notlimited to bacteria of the order Actinomycetales, also referred to asactinomycetes. Non-limiting examples of members belonging to the generaof Actinomycetes include Nocardia, Geodermatophilus, Actinoplanes,Micromonospora, Nocardioides, Saccharothrix, Amycolatopsis, Kutzneria,Saccharomonospora, Saccharopolyspora, Kitasatospora, Streptomyces,Microbispora, Streptosporangium, and Actinomadura. The taxonomy ofactinomycetes is complex and reference is made to Goodfellow,Suprageneric Classification of Actinomycetes (1989); Bergey's Manual ofSystematic Bacteriology, Vol. 4 (Williams and Wilkins, Baltimore, pp.2322-2339); and to Embley and Stackebrandt, “The molecular phylogeny andsystematics of the actinomycetes,” Annu. Rev. Microbiol. (1994)48:257-289, each of which is hereby incorporated by reference in itsentirety, for genera that may synthesize the compounds of the invention.

Preferably, a microorganism that is to be used for the fermentationproduction of ECO-4601 according to the process of the present inventionwill have inherent to the microorganism the genetic informationnecessary to allow for the microorganism to biosynthesize the compound.Preferably, the microorganism is a Micromonspora species, morepreferably Micromonspora sp 046-ECO11 a deposit of which was made onMar. 7, 2003, with the International Depository Authority of Canada(IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street,Winnipeg, Manitoba, Canada R3E,3R2, under Accession Number IDAC070303-01; more preferably Micromonspora sp [S01]046, a deposit of whichwas made on Dec. 23, 2003, with the International Depository Authorityof Canada (IDAC), Bureau of Microbiology, Health Canada, under AccessionNumber IDAC 231203-01; more preferably a Micromonospora strain that hasbeen selected for its ability to overproduce ECO-4601, and mostpreferably Micromonospora strain [S01U02]046, a deposit of which wasmade on Sep. 7, 2005, with the International Depository Authority ofCanada (IDAC), Bureau of Microbiology, Health Canada, 1015 ArlingtonStreet, Winnipeg, Manitoba, Canada R3E3R2, under Accession Number IDAC070905-01, and Micromonospora echinospora challisensis NRRL 12255 andStreptomyces carzinostaticus neocarzinostaticus ATCC 15944.

The deposit of the deposited strains has been made under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The deposited strainswill be irrevocably and without restriction or condition released to thepublic upon the issuance of a patent. The deposited strains are providedmerely as convenience to those skilled in the art and are not anadmission that a deposit is required for enablement, such as thatrequired under 35 U.S.C. §112. A license may be required to make, use orsell the deposited strains, and compounds derived therefrom, and no suchlicense is hereby granted.

Methods for generating strains of microorganisms capable ofbiosynthesizing increased levels of a particular bioactive compoundunder defined fermentation conditions, for example a compound thatpossesses an antibiotic activity or an anticancer activity, are known inthe art. Such methods may rely upon the selection of a mutant straingenerated as a result of a naturally-occurring mutation or by a mutationthat is induced due to treatment with a mutagenic agent. Examples ofsuitable mutagenic agents include, but are not limited to,N-methyl-N′-nitro-N-nitrosoguanidine, ethylmethansulfonate, nitrousacid, ultraviolet irradiation, X-rays and gamma irradiation, acombination of UV and psoralen, and transposon mutagenesis. Protocolsand parameters for the treatment of actinomycetes with such mutagenicagents are known in the art and can be found by reference to Manual ofIndustrial Microbiology and Biotechnology, 2^(nd) edition (1999),(edited by Demain A. L. and Davies J. E.) (American Society ofMicrobiology, Washington, D.C.) at pages 353-361 and 717-724. In regardsto the generation and selection of a mutant Actinomycete strain capableof overproducing a bioactive compound of interest, wherein the mutationevent that results in the overproduction phenotype occurs as anaturally-occurring event (i.e. a non-induced mutation), reference canbe made to Shima et al. (1996) “Induction of Actinorhodin Production byrpsL (Encoding Ribosomal Protein S12) Mutations That confer StreptomycinResistance in Streptomyces lividans and Streptomyces coelicolor A3(2)”Journal of Bacteriology, volume 178, pages 7276-7284, which describes amethod of obtaining strains of S. lividans and S. coelicolor thatproduce elevated levels of the antibacterial compound actinorhodin byscreening for naturally-occurring mutants that showed spontaneousresistance to the antibiotic streptomycin. Work performed subsequentlyby the Ochi et al. group (see Hesketh, A. and Ochi, K. (1997) “A NovelMethod for Improving Streptomyces coelicolor A3(2) for Production ofActinorhodin by Introduction of rpsL (Encoding Ribosomal Protein S12)Mutations conferring Resistance to Streptomycin”, J. Antibiotics, volume50, pages 532-535) disclosed a plausible link between the presence of acertain translation-associated mutations (alteration of the lysine aminoacid residue at position 88 in the ribosomal protein S12), which can beselected for by screening for Streptomyces strains resistant tostreptomycin, and the ability of the selected strains to biosynthesizelarger amounts of actinorhodin. A combination of methods may also beemployed to generate actinomycete strains capable of producing elevatedlevels of a bioactive compound of interest. In one embodiment, aMicromonospora strain of the present invention which is capable ofproducing an elevated level of the compound ECO-4601 in a fermentationculture was generated by exposing cells of a parent Micromonosporastrain to concentrations of the antibiotic streptomycin wherein theranges of concentrations used were greater than a minimal inhibitoryconcentration. An example of a minimal inhibitory concentration range ofstreptomycin is about 0.5 μg/mL to about 1.0 mg/mL. Preferably, aconcentration range of streptomycin utilized to select fornaturally-occurring resistance to the antibiotic is about 10 mg/ml toabout 100 mg/ml. More preferably, the concentration of the antibiotic isone that achieves a kill rate of at least 95%, and most preferably theconcentration of the antibiotic is one that achieves a kill rate of atleast 99%. A bacterial colony that remains viable upon exposure to alevel of streptomycin that provides a kill rate of at least 95% and morepreferably a kill rate of at least 99% is subjected to a furthermutagenesis treatment, for example, exposure to ultraviolet light.Levels of ultraviolet irradiation suitable for inducing mutations inActinomycetes are known in the art (see above, Manual of IndustrialMicrobiology and Biotechnology), and in one embodiment of the presentinvention treatment of a Micromonspora strain selected for itsresistance to high concentrations of streptomycin comprised exposure toultraviolet irradiation at an exposure level of about 40 Joles/m² toabout 45 Joles/m². In one embodiment of the present invention, suchultraviolet irradiation mutagenesis treatment results in aMicromonospora strain capable of an increased fermentation production ofECO-4601 of an at least five fold range when compared to thenon-ultraviolet irradiated strain from which a five fold-level producingstrain was derived.

ii) Recombinant Microorganisms

In another embodiment, the microorganism capable of producing thefarnesylated dibenzodiazepinone ECO-4601 may be provided by recombiningnucleic acid molecules that encode proteins useful in the production offarnesylated dibenzodiazepinones. Specifically, the microorganismcapable of producing the farnesylated dibenzodiazepinone ECO-4601 may beprovided by providing recombinant DNA vectors and nucleic acid moleculesthat encode all or part of the biosynthetic locus in strain 046-ECO11,which directs the production of ECO-04601, and is referred to herein as“046D”. Deposits of E. coli DH10B vectors, each harbouring a cosmidclone (designated as 046KM and 046KQ respectively) of a partialbiosynthetic locus for the farnesyl dibenzodiazepinone fromMicromonospora sp. strain 046-ECO11 and together spanning the fullbiosynthetic locus for production of ECO-04601 have been deposited withthe International Depositary Authority of Canada, Bureau ofMicrobiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba,Canada R3E,3R2 on Feb. 25, 2003. The cosmid clone designated 046KM wasassigned deposit accession numbers IDAC 250203-06, and the cosmid clonedesignated 046KQ was assigned deposit accession numbers IDAC 250203-07.

The deposit of the deposited E. coli DH10B vectors harbouring the cosmidclones has been made under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Micro-organisms for Purposesof Patent Procedure. The deposited clones will be irrevocably andwithout restriction or condition released to the public upon theissuance of a patent. The deposited clones are provided merely asconvenience to those skilled in the art and are not an admission that adeposit is required for enablement, such as that required under 35U.S.C. §112. A license may be required to make, use or sell thedeposited strains, and compounds derived therefrom, and no such licenseis hereby granted.

Further, the present invention may be provided by further including oneor more genetic modifications of 046D using conventional geneticrecombinant techniques, such as mutagenesis, inactivation, orreplacement of nucleic acids, to produce chemical variants of ECO-04601.

The farnesyl benzodiazepinone compound may be provided by a methodwherein a transformed host cell comprising a recombinant DNA vector thatencodes one or more of the polypeptides required for biosynthesis ofECO-4601, and culturing the host cell under conditions such thatfarnesyl benzodiazepinone is produced. In one embodiment, the host cellis a prokaryote. In another embodiment, the host cell is anActinomycete. In another embodiment, the host cell is a Streptomyceshost cell. In a further embodiment, the host cell is a non-StreptomyceteActinomycete such as a Rhodococcus, a Mycobacterium, or an Amycolatopsisspecies, or an non-Actinomycete bacterium such as a Brevibacterium or aspecies of Myxococcus such as Myxococcus xanthus (see Julien B. and ShahS. (2002) “Heterologous Expression of Epothilone Biosynthetic Genes inMyxococcus xanthus.” Antimicrobial Agents and Chemotherapy 46:2772-2778).

In a further embodiment, the present invention may be utilized inconjunction with recombinant nucleic acids that produce a variety offarnesyl dibenzodiazepinone compounds, or a dibenzodiazepinone bearingan acyl group other than a farnesyl moiety, that cannot be readilysynthesized by chemical methodology alone. The invention allows directmanipulation of 046D biosynthetic locus via genetic engineering of theenzymes involved in the biosynthesis of a farnesyl dibenzodiazepinone,or an analog thereof, according to the invention.

Farnesyl dibenzodazepinones and analogs can be produced by feeding oneor more key intermediates or biosynthetic precursors (as defined inFIGS. 1-4) or close structural analogs, to a host cell comprising arecombinant DNA vector that encodes one or more of the polypeptides ofthe present invention, and culturing the host cell under conditions suchthat the farnesyl benzodiazepinone or analog is produced. Keyintermediates are contacted directly with an isolated protein of theinvention to perform the necessary steps for the production of afarnesyl dibenzodiazepinone (e.g., the farnesyl diphopshate anddibenzodiazepinone precursors can be coupled using an IPTN protein ofthe invention).

Key intermediates may be commercially available or may be prepared usingstandard chemical procedures or using the proteins of this invention.For example, farnesyl diphosphate and 3-hydroxyanthranilic acid arecommercially available (e.g., Fluka F6892 and Aldrich 148776).3-Amino-5-hydroxybenzoic acid, a precursor of the2-amino-6-hydroxybenzoquinone, is prepared as described in Herlt et al(1981), Aust. J. Chem., volume 34, pages 1319-1324.

iii) Recombinant DNA Vectors

Vectors that may be used in conjunction with the present inventiontypically comprise the DNA of a transmissible agent, into which foreignDNA is inserted. A common way to insert one segment of DNA into anothersegment of DNA involves the use of specific enzymes called restrictionenzymes that cleave DNA at specific sites (specific groups ofnucleotides) called restriction sites. A “cassette” refers to a DNAcoding sequence or segment of DNA that encodes for an expression productthat can be inserted into a vector at defined restriction sites. Thecassette restriction sites are designed to ensure insertion of thecassette in the proper reading frame. Generally, a nucleic acid moleculethat encodes a protein useful in the production of a farnesyldibenzodiazepinone is inserted at one or more restriction sites of thevector DNA, and then is carried by the vector into a prokaryote e.g.Actinomycete, by transfection or transformation (see below). A segmentor sequence of DNA having inserted or added DNA, such as an expressionvector, can also be called a “DNA construct”. A common type of vector isa “plasmid” which generally is a self-contained molecule ofdouble-stranded DNA, usually of bacterial origin, that can readilyaccept additional (foreign) DNA and which can be readily introduced intoa suitable host cell. A plasmid vector often contains coding DNA andpromoter DNA and has one or more restriction sites suitable forinserting foreign DNA. A suitable vector system may also comprise aBacterial Artificial Chromosome (BAC) such as that described in Martinezet al. (2004), Applied and Environmental Microbiology, volume 70, pages2452-2463. Coding DNA is a DNA sequence that encodes a particular aminoacid sequence for a particular protein or enzyme. In one embodiment ofthe invention, the coding DNA encodes for polypeptides that may beuseful for the biosynthesis of a farnesyl dibenzodiazepinone.

Promoter DNA of a recombinant vector is a DNA sequence that initiates,regulates, or otherwise mediates or controls the expression of thecoding DNA. Promoter DNA and coding may be from the same or differentorganisms. Recombinant cloning vectors will often include one or morereplication systems for cloning or expression, one or more markers forselection in the host, e.g. antibiotic resistance, and one or moreexpression cassettes. Vector constructs may be produced usingconventional molecular biology and recombinant DNA techniques within theskill of the art. Such techniques are explained fully in the literature.See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A LaboratoryManual, Second Edition (1989) Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (herein “Sambrook et al., 1989”); DNA Cloning: APractical Approach, Volumes 1 and 11 (D. N. Glover ed. 1985); F. M.Ausubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley & Sons, Inc. (1994).

Examples of promoters that function in actinomycetes, e.g. Streptomyces,are taught in U.S. Pat. Nos. 5,830,695 and 5,466,590. Another example ofa transcription promoter useful in Actinomycetes expression vectors istipA, a promoter inducible by the antibiotic thiostrepton [c.f.Murakami, T., et al., (1989), J. Bacteriol, 171, 1459].

iv) Transformation of Actinomycetes

A suitable transformation method for use with an actinomycete comprisesforming the actinomycete culture into spheroplasts using lysozyme. Abuffer solution containing recombinant DNA vectors and polyethyleneglycol is then added, in order to introduce the vector into the hostcells, by using either of the methods of Thompson or Keiser [c.f.Thompson, C. J., et al., (1982), J. Bacteriol., 151, 668-677 or Keiser,T. et al. (2000), “Practical Streptomyces Genetics”, The John InnesFoundation, Norwich], for example. A thiostrepton-resistance gene isfrequently used as a selective marker in the transformation plasmid[c.f. Hopwood, D. A., et al., (1987), “Methods in Enzymology” 153, 116,Academic Press, New York], but the present invention is not limitedthereto. Additional methods for the transformation of actinomycetes aretaught in U.S. Pat. No. 5,393,665, and see also Martinez et al. (2004)(supra) which teaches transformation of Actinomycetes by conjugation.

Protocols for the transformation of Streptomyces and non-StreptomyceteActinomycetes are known in the art [see, for example, Dijkhuizen, L.“Genetics of Non-Streptomyces Actinomycetes” in Manual of IndustrialMicrobiology and Biotechnology, 2 edition, A. L. Demain and J. E. Davies(ed.) [1999, ASM Press, Washington, D.C.] at pages 368-378; and alsoNakashima N. et al. (2005) “Actinomycetes as host cell factories forproduction of recombinant proteins” Microbial Cell Factories 4:7].Shuttle-vectors which may be used for the cloning one or more genesinvolved in a farnesylated dibenzodiazepinone biosynthetic pathway intoa non-Streptomycete Actinomycete host. Such shuttle-vectors are made byrecombining, through known molecular genetic techniques, geneticsequences from one or more non-Streptomycete, for example Rhodococcus,plasmids DNA into an Escherichia coli plasmid or derivative plasmid togenerate a plasmid vector which is capable of replicating in both E.coli or Rhodococcus (while carrying the inserted gene(s) of interest).Such a host may include species of Rhodococcous, such as Rhodococcuserythropolis, Rhodococcus equi and Rhodococcus globerulus. Othernon-Streptomyces Actinomycete that may be transformed, using a shuttlevector specific to the particular genus of bacteria, and used forheterologous expression of one or more genes involved in biosynthesis ofthe farnesylated dibenzodiazepinone include species of the Mycobacterium(for example, Mycobacterium smegmatis), and species of the genusAmycolatopsis (for example, Amycolatopsis (Nocardia) lactamdurans and A.methanolica), and species of the genus Corynebacterium. Heterologousexpression of genes involved in biosynthesis of a secondary metabolitein a Micromonospora species has been successfully accomplished. Forexample, Saccharopolyspora erythraea (a megalomicin non-producer) wastransformed with Micromonospora megalomicea genes encoding forproduction of megosamine. Culture of the transformed S. erythraearesulted in the production of meaglomicin by the transformed host cells(see, Volchegursky Y. et al. (2000) “Biosynthesis of the anti-parasiticagent megalomicin: transformation of erythromycin to megalomicin inSaccharopolyspora erythaea” Molecular Microbiology 37: 752-762).

Use of an Actinomycete for heterologous expression of a gene clusterthat encodes for production of a microbial secondary metabolite that isused as an anti-tumor agent has been reported (see Sanchez C. et al.(2002) “The Biosynthetic Gene Cluster for the Antitumor Rebeccamycin:Characterization and Generation of Indolocarbazole Derivatives”Chemistry & Biology 9: 519-531; and Sánchez C. et al. (2005)“Combinatorial biosynthesis of antitumor indolocarbazole compounds”Proc. Natl. Acad. Sci. (U.S.A.) 102(2): 461-466). Furthermore, bacterialartificial chromosomes (BACs) for use in cloning very large fragments ofDNA (up to 100 kilobases) for transformation into Actinomycetes areknown in the art (see Sosio M. et al. (2000) “Artificial chromosomes forantibiotic-producing actinomycetes” Nature Biotechnology 18: 343-345),and which may be used to clone an entire gene cluster encoding forproduction of a microbial secondary metabolite or a natural bioanalogthereof.

v) Assay for Farnesyl Dibenzodiazepinone or Biosynthetic Intermediates

Actinomycetes defective in farnesyl dibenzodiazepinone biosynthesis maybe transformed with one or more expression vectors encoding one or moreproteins in the farnesyl benzodiazepinone biosynthetic pathway, thusrestoring farnesyl benzodiazepinone biosynthesis by geneticcomplementation (in cis or in trans) of the specific defect.

The presence or absence of farnesyl dibenzodiazepinone or intermediatesin the biosynthetic pathway (see FIGS. 1 to 4) in a recombinantactinomycete can be determined using methodologies that are well knownto persons of skill in the art. For example, ethyl acetate extracts offermentation media used for the culture of a recombinant actinomycetemay be generated, for example as described in Example 1, and fractionstherefrom containing farnesyl dibenzodiazepinone or intermediatesdetected by TLC on commercial Kieselgel 60F₂₅₄ plates. Farnesyldibenzodiazepinone and intermediate compounds are visualized byinspection of dried plates under UV light or by spraying the plates witha spray containing vanillin (0.75%) and concentrated sulfuric acid(1.5%, v/v) in ethanol and subsequently heating the plate. The exactidentity of the compounds separated by TLC is then determined usingliquid chromatography-mass spectroscopy. Methods of mass spectroscopyare taught in the published U.S. Patent Application Publication No.US2003/0052268.

vi) Mutagenesis

In a further embodiment, a microorganism capable of producing afarnesylated dibenzodiazeonone or analogs thereof, and which may be usedin conjunction with the present invention, may be provided for in partby direct manipulation of 046D biosynthetic locus via geneticengineering of the enzymes involved in the biosynthesis of the ECO-4601farnesyl benzodiazepinone.

A number of methods are known in the art that permit the random as wellas targeted mutation of the DNA sequences of the invention (see forexample, Ausubel et. al. Short Protocols in Molecular Biology (1995) 3rdEd. John Wiley & Sons, Inc.). In addition, there are a number ofcommercially available kits for site-directed mutagenesis, includingboth conventional and PCR-based methods. Examples include the EXSITE™PCR-Based Site-directed Mutagenesis Kit available from Stratagene(Catalog No. 200502) and the QUIKCHANGE™ Site-directed mutagenesis Kitfrom Stratagene (Catalog No. 200518), and the CHAMELEON® double-strandedSite-directed mutagenesis kit, also from Stratagene (Catalog No.200509).

In addition, mutations in the nucleotide sequence of the 046D locus maybe generated by insertional mutation or truncation (N-terminal, internalor C-terminal) according to methodology known to a person skilled in theart.

Older methods of site-directed mutagenesis known in the art rely onsub-cloning of the sequence to be mutated into a vector, such as an M13bacteriophage vector, that allows the isolation of single-stranded DNAtemplate. In these methods, one anneals a mutagenic primer (i.e., aprimer capable of annealing to the site to be mutated but bearing one ormore mismatched nucleotides at the site to be mutated) to thesingle-stranded template and then polymerizes the complement of thetemplate starting from the 3′ end of the mutagenic primer. The resultingduplexes are then transformed into host bacteria and plaques arescreened for the desired mutation.

More recently, site-directed mutagenesis has employed PCR methodologies,which have the advantage of not requiring a single-stranded template. Inaddition, methods have been developed that do not require sub-cloning.Several issues must be considered when PCR-based site-directedmutagenesis is performed. First, in these methods it is desirable toreduce the number of PCR cycles to prevent expansion of undesiredmutations introduced by the polymerase. Second, a selection must beemployed in order to reduce the number of non-mutated parental moleculespersisting in the reaction. Third, an extended-length PCR method ispreferred in order to allow the use of a single PCR primer set. Fourth,because of the non-template-dependent terminal extension activity ofsome thermostable polymerases, it is often necessary to incorporate anend-polishing step into the procedure prior to blunt-end ligation of thePCR-generated mutant product.

The protocol described below accommodates these considerations throughthe following steps. First, the template concentration used isapproximately 1000-fold higher than that used in conventional PCRreactions, allowing a reduction in the number of cycles from 25-30 downto 5-10 without dramatically reducing product yield. Second, therestriction endonuclease Dpn I (recognition target sequence: 5-Gm6ATC-3,where the A residue is methylated) is used to select against parentalDNA, since most common strains of E. coli Dam methylate their DNA at thesequence 5-GATC-3. Third, Taq Extender is used in the PCR mix in orderto increase the proportion of long (i.e., full plasmid length) PCRproducts. Finally, Pfu DNA polymerase is used to polish the ends of thePCR product prior to intramolecular ligation using T4 DNA ligase.

A non-limiting example for the isolation of mutant polynucleotides isdescribed in detail as follows:

Plasmid template DNA (approximately 0.5 pmole) is added to a PCRcocktail containing: 1× mutagenesis buffer (20 mM Tris HCl, pH 7.5; 8 mMMgCl2; 40 μg/ml BSA); 12-20 pmole of each primer (one of skill in theart may design a mutagenic primer as necessary, giving consideration tothose factors such as base composition, primer length and intendedbuffer salt concentrations that affect the annealing characteristics ofoligonucleotide primers; one primer must contain the desired mutation,and one (the same or the other) must contain a 5′ phosphate tofacilitate later ligation), 250 μM each dNTP, 2.5 U Taq DNA polymerase,and 2.5 U of Taq Extender (Available from Stratagene; See Nielson et al.(1994) Strategies 7: 27, and U.S. Pat. No. 5,556,772). Primers can beprepared using the triester method of Matteucci et al., 1981, J. Am.Chem. Soc. 103:3185-3191, incorporated herein by reference.Alternatively automated synthesis may be preferred, for example, on aBiosearch 8700 DNA Synthesizer using cyanoethyl phosphoramiditechemistry.

The PCR cycling is performed as follows: 1 cycle of 4 min at 94° C., 2min at 50° C. and 2 min at 72° C.; followed by 5-10 cycles of 1 min at94° C., 2 min at 54° C. and 1 min at 72° C. The parental template DNAand the linear, PCR-generated DNA incorporating the mutagenic primer aretreated with DpnI (10 U) and Pfu DNA polymerase (2.5 U). This results inthe DpnI digestion of the in vivo methylated parental template andhybrid DNA and the removal, by Pfu DNA polymerase, of thenon-template-directed Taq DNA polymerase-extended base(s) on the linearPCR product. The reaction is incubated at 37° C. for 30 min and thentransferred to 72° C. for an additional 30 min. Mutagenesis buffer (115ul of 1×) containing 0.5 mM ATP is added to the DpnI-digested, Pfu DNApolymerase-polished PCR products. The solution is mixed and 10 ul areremoved to a new microfuge tube and T4 DNA ligase (2-4 U) is added. Theligation is incubated for greater than 60 min at 37° C. Finally, thetreated solution is transformed into competent E. coli cells accordingto standard methods.

Methods of random mutagenesis, which will result in a panel of mutantsbearing one or more randomly situated mutations, exist in the art. Sucha panel of mutants may then be screened for those exhibiting reduceduracil detection activity relative to the wild-type polymerase (e.g., bymeasuring the incorporation of 10 nmoles of dNTPs into polymeric form in30 minutes in the presence of 200 μM dUTP and at the optimal temperaturefor a given DNA polymerase). An example of a method for randommutagenesis is the so-called “error-prone PCR method”. As the nameimplies, the method amplifies a given sequence under conditions in whichthe DNA polymerase does not support high fidelity incorporation. Theconditions encouraging error-prone incorporation for different DNApolymerases vary, however one skilled in the art may determine suchconditions for a given enzyme. A key variable for many DNA polymerasesin the fidelity of amplification is, for example, the type andconcentration of divalent metal ion in the buffer. The use of manganeseion and/or variation of the magnesium or manganese ion concentration maytherefore be applied to influence the error rate of the polymerase.

Genes for desired mutant polypeptides generated by mutagenesis may besequenced to identify the sites and number of mutations. For thosemutants comprising more than one mutation, the effect of a givenmutation may be evaluated by introduction of the identified mutation tothe wild-type gene by site-directed mutagenesis in isolation from theother mutations borne by the particular mutant. Screening assays of thesingle mutant thus produced will then allow the determination of theeffect of that mutation alone.

B. Culture Medium and Facilities

Microorganisms capable of producing ECO-4601 are cultivated in a culturemedium, preferably an aqueous culture medium, containing knownnutritional sources for actinomycetes. Such culture medium comprisesources of carbon, nitrogen, various inorganic salts and other nutrientsand growth factors that are assimable by actinomycetes. Preferredculture medium for fermentation of a microorganism capable of producinga farnesylated dibenzodiazepinone according to the present invention,preferably Micromonospora sp. [S01U02]046, include without limitationthe culture medium listed in Table 1, and other suitable culture mediuminclude, without limitation, the culture medium listed in Table 2. Forfermentation processes that are to be conducted on a large-volume scale,preparation of an inoculum seed culture of a microorganism capable ofproducing a farnesylated dibenzodiazepinone according to the presentinvention, preferably Micromonospora sp. [S01U02]046, can be performedusing culture medium KH, and culture medium HI can be used as theculture for the large volume fermentation. In another embodiment of thepresent invention, the seed medium is FBB (ingredient composition:potato dextrin (24 g/L), beef extract (3 g/L). Bacto-Casitone (5 g/L),glucose (5 g/L), yeast extract (5 g/L), and CaCO₃ (4 g/L, added afteradjusting medium to pH 7.0)). In a still further embodiment of thepresent invention, medium HI may be used as the seed medium. TABLE 1Examples of Preferred Culture medium Component per Culture medium (g/L)AP CA DZ GP HI JA MI PI QI QP YB pH 7 7 7 * 7 7.3 7 7 7 7.2 7 Glucose 6010 5 30 10 10 10 12 20 Cane 15 10 molasses Soluble starch 15 15 20 25Corn starch 30 Potato dextrin 40 20 40 10 50 Corn steep liquor 15 5 MOPS15 Yeast extract 8 8.34 5 5 5 1 Malt extract 20 35 Pharmamedia 30 15 1030 Glycerol 30 N-Z Amine A 10 Proflo Oil ™ 4 Fish meal 10 Bacto-peptone2.5 5 5 5 MgSO₄•7H₂O 1 CaCO₃ 2 5  7 3 2 2 2 3 5 NaCl 2 FeSO₄•7H₂O 0.01MgSO₄ 0.1 ZnSO₄•7H₂O 0.01 CoCl₂•2H₂O 0.001* pH not adjusted.

TABLE 2 Examples of Suitable Fermentation Media Component per Culturemedium¹ (g/L) AB AC CI DA FA HA KH MA MY QB RM VA VB ZL pH² * * 7.0 7.07.0 * 7.0 7.5 7.0 7.2 6.85 7.0 7.0 * Glucose 5 10 10 10 10 12 10 50 10 3Sucrose 340 100 20 Maltose 4 Cane molasses 10 15 20 Corn starch Solublestarch 25 15 5 25 10 Potato dextrin 20 20 40 20 10 Corn steep solid 5Corn steep liquor 10 5 Corn meal 10 Corn starch Soybean flour 5 30Soybean powder 15 Dried yeast 2 Torula yeast 4 Yeast extract 3 5 4 5Malt extract 3 10 Pharmamedia 10 Glycerol 25 20 10 Mannitol 25 N-Z AmineA 10 5 L-Glutamine 5.84 L-Arginine 1.46 Soybean powder Fish meal 10 5 10Bacto-peptone 5 Soytone-peptone 5 KH₂PO₄ 1 MgSO₄•7H₂O 0.5 0.5 1 1 CaCO₃4 2 3 2 1 4 6 2.5 5 NaCl 1 5 5 5 MnCl₂•4H₂O 0.1 ZnCl₂ 0.1 FeCl₂•4H₂O 0.1(NH₄)₂SO₄ 3.5 2 2 3 K₂SO₄ 0.25 MgCl₂•6H₂O 1 10.128 Na₂HPO₄ 3 2 KCl 2Phytic acid 1 Sodium citrate 2.5 Casamino acid 0.1 Proflo oil ™ (mL/L) 4MOPS 21 Trace element solution #1³ 2 (mL/L) Trace element solution #2⁴ 1(mL/L)¹Media components are described in g/L, unless otherwise noted.²The pH is adjusted as indicated prior to the addition of CaCO₃. Thesymbol * indicates no adjustment of pH.³Trace element solution #1 contains (per 100 mL of solution, made indeionized, distilled (dd) H₂O): ZnSO₄•7H₂O (10 mg), FeSO₄•7H₂O (100 mg),CuSO₄•5H₂O (10 mg), MnSO₄•H₂O (10 mg), Na₂B₄O₇•10H₂O (10 mg),(NH₄)₆MO₇O₂₄•4H₂O (10 mg).⁴Trace element solution #2 contains (per 1000 mL of solution, made indeionized, distilled (dd) H₂O): ZnCl₂ (40 mg), FeCl₂•6H₂O (200 mg),CuCl₂•2H₂O (10 mg), MnCl₂•4H₂O (10 mg), Na₂B₄O₇•10H₂O (10 mg),(NH₄)₆Mo₇O₂₄•4H₂O (10 mg).

Fermentation procedures for culturing a microorganism capable ofproducing a farnesylated dibenzodiazepinone according to the presentinvention follow equipment sterilization and safety practices andtechniques to allow for aseptic growth conditions pertaining to theculturing of microorganisms as practiced by those of skill in the art(see generally, Manual of Industrial Microbiology and Biotechnology,supra).

Suitable containers for fermentation of a culture medium inoculated witha microorganism capable of producing a farnesylated dibenzodiazepinoneaccording to the present invention are known in the art and can beselected depending on a volume of fermentation broth that is desired tobe produced. Containers for fermentation at a laboratory-scale level ofproduction may comprise, for example, the use of a sterile,foam-stoppered 2 litre, baffled Erlenmeyer type flask, containing about500 mL of culture medium and inoculated with a microorganism capable ofproducing a farnesylated dibenzodiazepinone. Containers containinginoculated culture medium can be agitated, for example by shaking on arotary shaker or in a heated water bath at a rotation speed of about 250revolutions per minute, to maintain a level of dissolved oxygen in themedia sufficient for the growth of the inoculated microorganism.Aeration may also be accomplished by injection of air, oxygen or anappropriate gaseous mixture into the culture medium during incubation. Asuitable fermentation temperature range and time to produce afermentation broth comprising the farnesylated dibenzodiazepinone, atsuch laboratory-scale level, are about 27° C. to about 29° C., and 72hours to about 100 hours, respectively. Suitable incubation conditionsalso comprise maintenance of the fermentation broth at a pH range ofabout 6 to about 9, and more preferably a pH range of about 7 to about8, during the incubation period.

In a research laboratory setting, an automated benchtop fermentor may beutilized to produce a quantity of a fermentation broth comprising thefarnesylated dibenzodiazepinone. Such fermentors are known to those ofskill in the art and are available from a number of commercialsuppliers, for example New Brunswick Scientific Inc. or Sartorius AG.Such fermentors may provide a range of fermentation volumes, for examplefrom about 1 liter to about 20 liters, with a working volume in a rangeof less than one liter to about 15 liters. Preferably, such a fermentoris equipped with systems and accessories for control and monitoring offermentation conditions, for example: a heat-blanketed or water-jacketedstainless steel or glass fermentation vessel or a vessel having animmersed heating/cooling coil; a mechanically-sealed stirring shaftfitted with one or more suitable impellers (for example, Rushton-typeimpellers) and which can be operated at variable rotational speedssuitable to achieve proper agitation of the fermentation broth; one ormore peristaltic pumps for the addition or removal of liquids; one ormore probes to automate and monitor liquid additions/removals andcontrol of foaming of the fermentation broth; probes and controls formonitoring and adjusting of the fermentation broth pH and dissolvedoxygen content; solenoid valves to allow for a sequential addition ofdesired gases to the fermentation broth; and an electronic control unitequipped (which may include a visual display system) with appropriatesoftware for programming and monitoring of the fermentation conditionsand which may provide an electronic and/or printed record of thefermentation. In a preferred embodiment of the present invention, a NewBrunswick Scientific Inc. BioFlo® 110 fermentor is used for fermenting astrain of a microorganism capable of producing ECO-4601.

In a further embodiment of the present invention, fermentation of astrain of a microorganism capable of producing ECO-4601 can occur usinga large volume fermentor, more preferably using one or more pilot plantscale fermentation vessels. Pilot plant scale vessels, as recognized bythose of skill in the art, may range in size of their volumetriccapacity from about 20 litres to about 7,500 litres, more preferablyfrom about 75 litres to about 3,000 litre vessels, and even morepreferably from about 100 litre to about 2,000 litre vessels. Numerousmanufacturers and suppliers of large-scale fermentation vessels andaccessory systems are available for those of skill in the art. Suchsuppliers and manufacturers may include, but are not limited to, NewBrunswick Scientific Co. (Edison, N.J.), Chemap AG (Volketswil,Switzerland), Applikon Inc. (Schiedam, The Netherlands), Sartorius BBISystems, Inc. (Bethlehem, Pa.), Marubishi Laboratory Equipment Co., Ltd.(Japan), and L.H. Engineering Co. (United Kingdom). As noted byHosobuchi M. and Yoshikawa “Scale-Up of Microbial Processes” (in Manualof Industrial Microbiology and Biotechnology, 2^(nd) edition (1999),(edited by Demain A. L. and Davies J. E.) (American Society ofMicrobiology, Washington, D.C.) at pages 353-361) and by Junker B. in“Multipurpose Fermentor Design: Critical Considerations” ChemicalEngineering February 2003 at pages 1-6, a number of factors may requireadjustment in order to optimize conditions for large-scale fermentationproduction depending upon the size and type of fermentation vesselselected for use. Such factors may include, for example, those of amechanical/physical nature such as the type and number of impellers andbaffles, the agitation speed and power per unit volume, the impeller tipspeed (which may influence shear stress inflicted upon themicroorganisms), and the aeration rate and pressure. As will beappreciated by those of skill in the art, depending upon the capacity ofthe large volume fermentation vessel that is to be used, inoculation ofthe culture medium in the selected large volume fermentation vessel mayrequire as fermentation production of one or more seed cultures of themicroorganism capable of producing the farnesylated dibenzodiazepinonewhich are subsequently utilized for production of a quantity of inoculumsufficient for inoculation of the culture medium in the large volumefermentation vessel. Seed cultures may be prepared, for example, in oneor more baffled seed flasks maintained as per conditions forfermentation production at a laboratory-scale level of production, andone or more aliquots from such seed cultures may be withdrawn during toaccess purity and viability of the culture prior to inoculation of theinoculum fermentor. Production of a volume of inoculum fermentationbroth should be commensurate with the volume of culture medium to beinoculated in the large fermentation vessel. Preferred volume range ofan inoculum fermentation broth for use in accordance with the presentinvention is about 2% to about 10% of the volume of the volume ofculture medium contained in the large volume fermentation vessel, andmore preferably about 3% to about 4% of the volume of the culture mediumcontained in the large volume fermentation vessel, and most preferablyabout 3.2% to about 3.5% of the volume of the culture medium containedin the large volume fermentation vessel. In accordance with the presentinvention, a Chemap AG type M16K inoculum fermentor may be used forproducing an inoculum culture that can be used to inoculate a volume ofculture medium contained in a large volume fermentor. A Chemap AG typeCF/569 having a total capacity of 750 litres with impellers mounted on adrive (as measured from the bottom of the vessel) and situated at aabout a 70 litre mark, and at about a 200 litre mark, and at about a 340litre mark may be used in accordance with the present invention.

It will be recognized by those of skill in the art that the firstconcentrate comprising the farnesylated dibenzodiazepinone of Formula Iobtained by the method of the present invention may be processeddownstream of the production of the first concentrate so as to reduce alevel of an impurity in the first concentrate. Such processing may occurin any one of a number of manners as recognized by those of skill in theart, for example, by employing a column cleanup process to reduce alevel of an impurity, as exemplified in one or more of the examplesbelow which are not to be considered as to be limiting as to aparticular method that may be selected so effect such downstreamprocessing.

EXAMPLES

Unless otherwise indicated, reagents and solvents used in the followingexamples were supplied by Sigma-Aldrich and or Fisher Scientific. Unlessotherwise indicated, all numbers expressing quantities of ingredientsand properties used in the specification and claims are to be understoodas being modified in all instances by the term “about”. Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe present specification and attached claims are approximations. At thevery least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalparameter should at least be construed in light of the number ofsignificant figures and by applying ordinary rounding techniques.Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set in the examples, Tables and Figures are reported as preciselyas possible. Any numerical values may inherently contain certain errorsresulting from variations in experiments, testing measurements,statistical analyses and such.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

Example 1 Production of Micromonospora sp. Strain [S01U02]

1.1 First Round of Strain Improvement

Micromonospora sp. strain 046 (accession number IDAC 070303-01) wasstreaked on culture plates bearing GYM agar medium containing 10-100μg/mL streptomycin. The ingredients for GYM agar medium comprised (ing/L): glucose (4 g), yeast extract (4 g), malt extract (10 g), N-Z AmineA (1 g), NaCl (2 g), agar (20 g), made to 1000 mL with distilled water,pH 7.2 (see Shima J. et al. (1996) “Induction of Actinorhodin Productionby rpsL (Encoding Ribosomal Protein S12) Mutations that ConferResistance in Streptomyces lividans and Streptomyces coelicolor A3(2)”J. Bacteriol., vol. 178 (24), pages 7276-7284). Inoculated plates wereincubated at 28° C. for 4 to 10 days. Single colonies exhibiting naturalresistance to the antibiotic were obtained, and were thereafter streakedonto GYM agar media plates containing a similar concentration ofstreptomycin as the plate from which the particular colony was selected.The plates were incubated at 28° C. for 15 to 20 days. Surface growthfrom each plate was used to inoculate (for each plate) one glass culturetube (150 mm×20 mm) containing 5 mL medium KH (no streptomycin was addedto the seed medium). Inoculated culture tubes were incubated at 28° C.for a period of 70-72 h on a rotary shaker operating at 250 RPM to allowfor bacterial cultures to grow. After the cultivation period, a 0.250 mLaliquot was taken form each culture tube (seed culture) and wastransferred into similar tubes containing 10 mL culture medium CI.

Inoculated culture tubes were incubated at 28° C. for a period of 7 dayson a rotary shaker operating at 250 RPM to allow for bacterial culturesto grow and produce ECO-4601. After completion of the fermentation, 10mL of ethyl acetate was added to each tube, and the tubes were returnedto the shakers for agitation for 20 min. The content of each tube wastransferred into separate 45 mL Falcon tubes and centrifuged at 4500 RPMfor 10 min to generate an upper organic phase and a lower aqueous phasewithin the tube. A 5 mL aliquot of the organic phase (upper phase) wastaken from each tube and transferred into separate small glass tubes(100×13 mm), and the solvent was evaporated under nitrogen stream togive a dried residue within each tube. The dried residue wasre-dissolved in 0.5 mL methanol and assayed by LCMS for ECO-4601content. Approximately 850 single colonies were screened as such, fromwhich a single colony (clone) was identified that produced ECO-4601 at ayield of approximately 10-25 mg/L, such yield being approximately 10fold higher than that produced by controls (equivalent liquid culturesof the parent strain). The selected clone was resistant to the highestconcentration of streptomycin (100 μg/mL), and was designatedMicromonospora sp. strain [S01]046 (accession number IDAC 231203-01).

1.2 Second Round of Strain Improvement

In a second round of strain improvement, Micromonospora sp. strain[S01]046 was streaked on culture plates bearing GYM agar medium. Theplates were incubated at 28° C. for 15-20 days until abundantsporulation occurred. Spores were collected from each plate by floodingthe plate with 5 mL of sterile distilled water, scraping off the sporesfrom the entire plate and pipetting the suspended spores into a 15 mLconical tube that was then centrifuged at 5000 RPM for 10 minutes at 4°C. The supernatant was discarded by pipetting off from the centrifugedtube, and the spore pellet resuspended in 10 mL of sterile distilledwater and thoroughly vortexed. Agar pieces, if present, were removedfrom the spore resuspension by filtering the resuspension through cottonplaced in a syringe followed by re-centrifugation at 5000 RPM for 10minutes at room temperature, followed by decanting of the supernatant bypipetting-off. Centrifuged spores resuspended with sterile distilledwater and diluted (with sterile distilled water) to reach a level of8000 to 20000 spores/mL.

From the diluted spore suspension (whether cotton-filtered or not), a0.5 mL aliquot was taken and drops were evenly spread onto the agarsurface of GYM agar plates. Plates were allowed to dry in laminar flowunder aseptic conditions for 10 minutes, while the lid of each playedwas kept ajar during the drying period. The plated spores were thensubjected to ultraviolet (UV) irradiation at intensities of 40-45Joles/m² of the agar surface. The lids were removed from the platesduring the UV treatment. To prevent reversal, by light energy, ofmutations induced by the UV treatment, plates that were subjected to theUV treatment were thereafter maintained in a light-proof container at28° C. for at least 24 hours. The irradiated plates were thereafterincubated at 28° C. for 4 to 10 days or until single colonies appeared.Surviving colonies (approximately 0.1%) were picked, streaked onto GYMagar media plates and incubated at 28° C. for 15 to 20 days or untilabundant sporulation occurred.

Clones from the surviving colonies were screened for production ofECO-4601 as described above for first round screening to obtainantibiotic resistant clones. Non-UV treated cultures of strain [S01]046were grown under similar conditions and used as control. From thissecond round of screening, a single strain was identified to produceECO-4601 at 4-6 fold higher than the control strain. This new strain wasdesignated Micromonospora sp. strain [S01U02]046 (deposit accessionnumber IDAC 070905-01). When grown in 14-L fermentors containing 10 L ofculture medium HI (Table 1), strain [S01U02]046 was found to produceECO-4601 at a yield rate of approximately 172±24 mg/L.

Example 2 Small-Scale Laboratory Production

To assess whether a variation in the fermentation conditions may resultin an increase in yield of ECO-4601 in the fermentation broth, asmall-scale fermentation and extraction procedure was adopted asfollows.

2.1 Preparation of Flasks

Three glass beads (5 mm diameter) were placed in each of two, 125 mLErlenmeyer flasks and the flasks are autoclaved. A volume of 25 mL ofsterilized HI media was then aseptically transferred into each flask.Thereafter, an aliquot of conditioned, sterile HP20 resin suspension wasadded to each flask to give a 1% HP20 resin content per flask (1 mL froma 0.25 g/mL sterile stock solution). Preparation of the HP20 resinsuspension consisted of measuring an amount of dry HP20 into a beakerand adding a volume of 100% methanol sufficient to cover the dry resin.The contents of the beaker were then thoroughly mixed to evacuate anyair bubbles from the resin, and the HP20 resin was allowed to settle inthe beaker. After settling, the methanol was decanted, and the methanoltreatment step repeated once. The methanol-conditioned HP20 resin wasthen washed 5 times with distilled water, and after the fifth wash,sufficient water was added to the sterile conditioned HP20 resin to givea stock solution having a concentration of 0.25 g/mL, and the preparedflask sterilized in an autoclave (121° C.) for 30 min.

2.2 Preparation of Seed Culture

A seed culture of Micromonospora sp. strain [S01U02]046 was prepared bytransferring the contents of two glycerol stock vials (each vial wasmaintained at −80° C. until use and contained 1.5 mL of frozen bacterialculture) into one, 2-L baffled flask containing 500 mL of aqueousculture medium KH. The seed culture was grown at 28° C. for 3 days withshaking at 250 RPM.

To produce the glycerol stock tubes, surface growth from a sporulatedGYM plate of [S01U02]046 that was incubated at 28° C. for 17 to 20 dayswas transferred into three, 2-L baffled flasks each containing 500 mL ofKH medium. The cultures were then grown at 28° C. for 3 days withagitation at 250 RPM. A pooled culture was prepared by mixing 100 mLfrom each flask in a separate sterile flask (300 mL total) under asepticconditions. An equal volume from a sterile 40% (v/V) glycerol-in-watersolution was added to the pooled culture, mixed and dispensed in 1.5 mLaliquots in 2 mL screw cap tubes and stored at −80° C.

2.3 Inoculation of Production Culture Medium

For each experiment, duplicate Erlenmeyer flasks were prepared as perSection 2.1, and each flask was inoculated with a 0.5 mL (2% V/V)aliquot of the seed culture grown as described above at 250 RPM.Inoculated flasks are placed in a rotary shaker that is operated at 250RPM and cultures are grown at 28° C. for a period of 4 days.

2.4 Extraction

On the fourth day (96 hrs) of fermentation culturing, a 2 mL aliquot ofthe fermentation broth was removed from each flask and transferred intoa 15 mL screw cap tube to which 2 mL of ethyl acetate was added. Thetube was then vortexed at high speed for approximately 2 minutes, andthen centrifuged (Sorval Legend™ RT) for 10 minutes at 5350 RPM at roomtemperature to generate an upper organic phase and a lower aqueous phase(containing bacterial mycelia and the HP20 resin) within the tube. A 0.5mL aliquot of the upper was removed and transferred to a borosilicateglass tube (13×100 mm) and the contents of the glass tube dried undernitrogen at 40° C. for 10 minutes. The resulting dried residue wasresuspended in 0.5 mL of 100% methanol (with brief vortexing to ensurecomplete resuspension). A 150 μL aliquot of the residue resuspension wastaken for analysis of ECO-4601 content.

2.4 Results

Results from replicate experiments to assess whether the presence of anamount of methanol-conditioned HP20 resin in the fermentation culturebroth during fermentation period resulted in an increased production ofECO-4601 are shown in Table 3 directly below. TABLE 3 Small-scalefermentation broth concentrations of ECO-4601 ECO-4601 CONTENT (mg/mL offermentation broth, based on analysis of extracts of the fermentationbroth at 96 hrs) Experiment 1 Experiment 2 CONDITION Flask 1 Flask 2Flask 1 Flask 2 HI culture medium 7.00 7.30 7.36 7.21 HI culturemedium + 329 302 263 265 HP20 (1% in media)

The above-noted results indicated that the presence of resin in thefermentation broth could correlate with an increase in production ofECO-4601 and thereby result in an increased ECO-4601 yield.

Example 3 Small Scale Laboratory Production

A small-scale fermentation culture was grown and extracted as describedabove in Example 2 with the exception that triplicate flasks were grownfor a single experiment. The results from the triplicate flasks areshown below in Table 4. TABLE 4 Small-scale fermentation brothconcentrations of ECO-4601 ECO-4601 CONTENT (mg/mL of fermentationbroth, based on analysis of extracts of the fermentation broth at 96hrs) Experiment 1 CONDITION Flask 1 Flask 2 Flask 3 HI culture medium 77.33 8.01 HI culture medium + HP20 465 310 380 (1% in media)

The above-noted results further indicated that the presence of resin inthe fermentation broth could correlate with an increase in production ofECO-4601 and thereby result in an increased ECO-4601 yield.

Example 4 Scale-up Production—Benchtop Fermentation Batch Cultures

4.1 Inoculum Production and Benchtop Fermentation

Micromonospora sp. strain [S01U02] (deposit accession number IDAC070905-01) was maintained on agar plates of GYM agar. An inoculum forthe benchtop fermentation was prepared by transferring the surfacegrowth of the Micromonospora sp. from the agar plates to 2-L flaskscontaining 500 mL of sterile KH medium. Each liter of KH mediumcomprises 10 g glucose, 20 g potato dextrin, 5 g yeast extract, 5 gNZ-Amine A, and 1 g CaCO₃ made up to one liter with water (pH 7.0). Theinoculum culture was incubated at about 28° C. for approximately 70hours on a rotary shaker set at 250 rpm. Following incubation, 300 mL ofculture was transferred to a 14.5 L BioFlo® 110 fermentor (New BrunswickScientific, Edison, N.J.) containing 10 L of sterile production culturemedium HI. Each liter of production medium HI was composed of 20 gpotato dextrin, 30 g glycerol, 2.5 g Bacto-peptone, 8.34 g yeastextract, and 3 g CaCO₃, (added after pH adjustment) (with 0.3 mLSilicone defoamer oil (Chem Service) and 0.05 ml Proflo oil™ (Tradersprotein) as antifoam agents). Media contents were mixed into 3 L ofdistilled water for approximately 30 minutes using a magnetic stirreroperating at a medium speed, and the contents adjusted to pH 7.0followed by the addition of the CaCO₃, Proflo™ oil and Silicon defoamer.The vessel was sterilized at 121° C. for 40 min. Cultures were grown byfermentation for 96±4 hr at 28° C., with dissolved oxygen (DO)controlled at 25% in a cascade loop with agitation varied between350-650 RPM and aeration set at a fixed rate of 0.5 V/V/M.

To assay for the amount of ECO-4601 present in the fermentation broth atthe time of completion of fermentation period, a 5 mL aliquot of thefermentation broth was also removed (in-process fermentation samplingand extractions were also performed on the morning and evening of days 2and 3 for each fermentation culture). The 5 mL aliquot was subjected tothe ethyl acetate extraction procedure as generally described above inExample 2, section 2.3, with the exception that the 5 mL aliquot of thefermentation broth was extracted with 5 mL of ethyl acetate in a 50 mLscrew cap tube, and that the dried residue was resuspended in 1 mL of100% methanol. The methanol-resuspended residue was thus diluted by afactor of 2× for assay by HPLC/UV to determine the production yield(concentration expressed as mg/L) of ECO-4601 in the fermentation broth.HPLC/UV assays were performed using either a Waters Alliance 2690Separations Module (Waters Corp., Milford, Mass.) equipped with a WatersPDA 996 detector, or a Waters Alliance 2695 Separations Module equippedwith a Waters Dual Wavelength 2487 detector. HPLC columns used wereselected from either an ACE 3 AQ C18, 3.0×100 mm+3.0×10 mm guard 3 u(Canadian Life Sciences, Inc., Peterborough, Ontario, Canada) or aSymmetry C18, 4.6×150 mm+3.0×20 mm guard 5 u (Water Corp.). For bothcolumn types, the solvents used were: Solvent A—H₂O+0.4% formic acid(996:4 mL); Solvent B—Acetonitrile+0.4% formic acid (996:4 mL). Theflowrate and time for the ACE column was set to 1.00 mL/min and 16minutes, while the flow rate for the Symmetry column was set 1.30 mL/minand 15 minutes. For both column types, a 20 μL sample was injected atroom temperature, with pressure set at a minimum of 20 bar and a maximumof 300 bar, with detection occurring at 294. Optionally, detection couldhave been performed at 230 nm.

A number of separate fermentation broth cultures were grown as describedabove. As noted above, in-process samples aliquots were taken from eachfermentation broth on the morning and evening of the second day offermentation; on the morning, afternoon and evening of the third day offermentation; and on the morning of the fourth day of fermentation. Foreach fermentation culture, a yield rate (that being a rate of productionof ECO-4601 in terms of mg of ECO-4601 per liter of broth per hour)could be assessed given an overall time period for each fermentationculture and mass of ECO-4601 present in the fermentation broth at thetime of completion of the fermentation. Also, it was observed that foreach fermentation culture the rate of production of ECO-4601 varied overa range of values throughout the course of the fermentation culture timeperiod, but increased more steeply between the second and third day offermentation culturing. Values for the yield rate for the series offermentation cultures noted directly, calculated on the basis ofin-process sample aliquots taken on the afternoon of the second day andthe morning of the third day of fermentation are presented in Table 5,noted directly below. TABLE 5 In-process Fermentation Culture AnalysesBioFlo 110 Fermentation Broth Batch No. 135 136 137 138 139 140 141 142Day 2 PM 24.16 7.26 30.6 28 54.6 18.05 14.06 11.21 [ECO-4601 mg/L] Day 3AM 93.49 60.24 128.4 126.8 130 73.63 64.77 47.72 [ECO-4601 mg/L] Numberof 17 17 19.5 19.5 19.5 17 17 17 Hours b/n D2-PM & D3-AM SamplesDifference in 69.33 52.98 97.8 98.8 75.4 55.58 50.71 36.51 [ECO-4601]b/n D2-PM & D3-AM Yield Rate 4.08 3.12 5.01 5.06 3.87 3.27 2.95 2.15(mg/L/hr)

Results shown in Table 5 indicated that while the yield rate can varybetween fermentation batches, a yield rate of over 5 mg/Uh was achieved.

Presented below in Table 6, from each of the separate fermentation brothcultures grown as described above, are the data which showed ECO-4601fermentation broth concentration and mass per fermentation broth cultureat the time of completion of each fermentation broth culture'sincubation period. TABLE 6 Fermentation broth concentrations of ECO-4601(on completion of fermentation) BioFlo 110 Fermentation Broth Batch No.135 136 137 138 139 140 141 142 Broth 97.56 88.71 122 137.6 130.4 108.8754.49 115.08 concentration of ECO-4601 (mg/L) Mass (mg) of 878 798.41098 1238.4 1173.6 1088.7 517.7 1093.3 ECO-4601 in Broth

4.2 Harvesting, Extraction and Concentration

For each fermentation batch culture, after removal of the assay aliquotfrom the fermentation broth, the pH of the fermentation broth wasadjusted to 3.0±0.2 by the drop-wise addition of 20% aqueous H₂SO₄(sulfuric acid) and with constant stirring. The resulting acidifiedmixture was further treated by being cooled to 4° C. and incubated atthat temperature for a period of 12 h to 48 h (maximum 72 hours).Batches were combined into larger holding flasks or carboys (10 L or 20L volume, as required) for the period at which they were held at 4° C.On completion of the 4° C. incubation period, the cold fermentationbroth was aliquoted into 750 mL polypropylene centrifuge tubes andcentrifuged (Sorval RT-7) at 3000-3500 rpm for 15-20 min. Thesupernatant was discarded and the mycelial pellets were combined to givea total combined pellet mass of 1400 grams. The combined mycelial pelletwas then extracted three times (90 minutes, 90 minutes, 30 minutes, withstirring at medium to high speed) with 100% methanol (HPLC grade, J. T.Baker), using a methanol volume per extraction round of 300±5 mL forevery 100 g of mycelia. The methanol extracts (three rounds) were pooledand filtered through Whatman #4 filter paper.

The filtered extract was treated, as follows, to form a firstconcentrate: 330 g of HP20 resin was loaded into a 10 L rotaryevaporator flask, which was then mounted onto a rotary evaporator system(Buchi Rotavapor® (Model R-200) equipped with a Buchi heating bath(Model B-490) set to 40° C., and a Buchi vacuum pump (Model V-500)).Aliquots of the filtered extract were introduced into the evaporatorflask over the course of an evaporation process that lasted 22±2 hoursevaporation. On completion of the evaporation process, a volume of 2200mL of residual water remained in the evaporation flask together with theHP20 onto which the ECO-4601 was adsorbed. HPLC-UV analysis of theresidual water indicated that 69 mg of ECO-4601 remained in the residualwater (31.36 mg/L), thereby indicating that over 99% of the ECO-4601from the filtered extract was adsorbed onto the HP20 resin.

Example 5 Scale-up Production—Benchtop Fermentation Batch Cultures

In a further example of a benchtop fermentation, fermentation cultureswere generated, as described in Example 4, using the BioFlo® 110fermentor. Aliquots from each fermentation batch were taken upon thecompletion of the fermentation period for each batch and analyzed byHPLC-UV as described in Example 4 to establish the ECO-4601concentration, and thereby the mass of ECO-4601, in each fermentationbatch culture at the time of the fermentation period's completion perculture. Results from such analyses are shown below in Table 7. TABLE 7Fermentation broth concentrations of ECO-4601 (on completion offermentation) BioFlo 110 Fermentation Broth Batch No. 4001 4001 x02 x034002 105 106 107 108 109 Concentration of 101.56 139 90.43 152 91 117157 122 ECO-4601 (mg/L) ECO-4601 810.24 1320.5 723.44 1216 864.5 1111.51491.5 1159 mass (mg) in Broth

Upon completion of the fermentation period, the fermentation cultureswere acidified, combined (total volume of the combined fermentationbroths being about 71.5 L) and cooled as described above prior toharvesting of the particulate matter (mycelia) from the combinedfermentation broths by centrifugation. Harvested mycelia were subjectedto 3 rounds of methanol extraction as described in Example 4. HPLC-UVanalysis of the methanol extract indicated an extracted mass of ECO-4601of 7470 mg.

Example 6 Scale-up Production—Large Volume/Pilot Plant Fermentation

6.1. Preparation of a Master Seed Stock

Pilot manufacturing master seed stocks were prepared as generallyillustrated in FIG. 5. A spore pellet was obtained from a lyophilizedmaster vial of Micromonospora sp. [S01U02]046, and was asepticallytransferred into a sterile, 50 mL tube to which 2.5 mL of TSB-medium(Trypticase Soy Broth, from Becton Dickson) had been added. The tube wasthen vortexed to obtain a homogeneous suspension of the lyophilizedspore pellet. Two 0.5 mL aliquots of the pellet suspension werewithdrawn from the tube and spread evenly on separate GYM agar controlplates (GYM agar medium (per 1000 mL): glucose (4 g); yeast extract (4g); malt extract (10 g); NZ-Amine A (1 g); NaCl (2 g); agar (20 g), pH7.2) to assess for purity and growth of the bacteria. Inoculated GYMplates were incubated for at 28±1° C. for about 72 hours. Concomitantwith the inoculation of the GYM control plates, a 0.5 mL aliquot of thepellet suspension was inoculated into each of two 125 mL flasks eachcontaining 25 mL of KH media and three glass beads (5 mm diameter).Inoculated flasks were incubated at 28±1° C. for about 72 hours to about96 hours in a rotary shaker operating at 250-300 RPM (until orangemycelia appeared in the culture medium). Aliquots from the 125 mL flaskswere then plated on GYM agar plates and incubated for 17.5±2.5 days toobtain dark spores at the mycelial surface. Spores were collected (in alaminar flow hood) by flooding each plate with about 5 mL of steriledistilled water and scraping with a sterile bacteriological loop, flowedby transfer of the resuspended spores into a 50 mL conical tube viafiltering through cotton plugs. The spore suspension was thencentrifuged (5,000 RPM, 10 minutes at 4° C.) to obtain a spore pellet,which was subsequently washed (vortexed) with 10 mL of sterile water andre-centrifuged (5,000 RPM, 10 minutes at 4° C.). The final spore pelletwas then resuspended in a 12% skimmed milk suspension (Sigma) in waterto a homogenous suspension and then aseptically dispensed in 0.5 mLaliquots into lyophilization vials. Vials were then frozen and followedby lyophilization, and the lyophilized master seed stock vials were thensealed under vacuum and stored at room temperature or for longer termstorage maintained at 5±3° C. in a refrigerator.

6.2. Preparation of a Working Stock

Preparation of a working stock for pilot-scale fermentationmanufacturing followed a procedure as generally illustrated in FIG. 5(lower portion). The contents of a lyophilized master stock vial wereaseptically transferred to a KH media-containing tube and suspended byvortexing. The suspended pellet was transferred to flasks containing KHmedia and glass beads, which are incubated in an orbital shaker for84±12 hours, as described in section A above. Contents of the cultureflasks were subsequently transferred onto GYM agar plates and incubatedas described above. Spores when then collected as described above,washed and aseptically transferred to a tube and centrifuged asdescribed above. Following centrifugation, the pellet was resuspended inwater, filtered, re-centrifuged and the supernatant removed. The sporepellet was then subsequently resuspended in a 20:80 glycerol:watersolution, vortexed and aliquoted in vials as working stocks to be usedfor generation of pilot-scale fermentation cultures. Working stockaliquots are stored at −70±10° C.

6.3. Preparation of Working Seed Plates

A working stock vial was retrieved from storage and maintained on dryice. GYM agar plates where thereafter streaked using a bacteriologicalloop containing a working stock scraped from the vial with the loop.Streaked plates were inverted and incubated for about 15 to about 20days at 28±1.0° C. to obtain a bacterial lawn with dark, waxy spores onthe surface.

6.4. Incubation of the Seed Flasks

Preparation of seed flasks for pilot-scale fermentation manufacturingfollowed a procedure as generally illustrated in FIG. 6 (upper portion).Surface growth, comprising cell mass and spores, from each GYM agarplate was transferred to three, 2 L baffled flasks containing 500 mL ofsterile KH medium per flask, and the inoculated flasks were incubatedfor 70 to 72 hours at 28±1.0° C. on an orbital shaker operating at about300 RPM to produce seed cultures. Purity and viability of the seedcultures was verified by in-process testing consisting of microscopicexamination of aliquots taken from the growing cultures at about 48hours and at about 72 hours post-inoculation of the flasks, and also bystreaking GYM agar at these point-inoculation timepoints.

6.5. Fermentation of Inoculum Fermentor

Culture material from the three seed flasks was pooled (FIG. 6, lowerportion), and under aseptic conditions, a volume (450 mL) of the pooledmaterial equivalent to about 3% of a volume of culture medium present ina 28 L capacity inoculum fermentor (Chemap AG, Type M16K) wastransferred aseptically to the inoculum fermentor containing 15 L of KHaqueous culture medium at pH 7. Culture medium was supplemented with 3mL of Silicone defoamer oil (Chem Service) and 0.75 mL of Proflo oil(Trader Protein; Southern Cotton Oil Co.). Fermentation was performed at28±1.0° C. for 48±1 hours, with dissolved oxygen maintained at 25%linked to agitation and aeration at 0.55 (V/V/M), and a mixing speed of110 to 400 RPM. In-process testing assay for purity and viability of theinoculum fermentor culture consisted of microscopic examination andstreaking of 24 hour post-inoculation and 48 hour post-inoculationaliquots on GYM agar plates.

6.6. Fermentation in Pilot Fermentor

The entire volume from the inoculum fermentor (FIG. 6, lower portion)was transferred to a 750 L capacity pilot fermentor (Chemap AG, typeCF/569) containing 450 L of HI culture medium adjusted to a pH of 7.1.The inoculum volume that was transferred corresponded to about 3.3% ofthe volume of medium in the pilot fermentor. Silicone defoamer oil (135mL) and Proflo oil (22.5 mL) were also added to the culture mediumbefore sterilization at 121° C. for 40 minutes. Fermentation wasperformed at 28±1.0° C. for 95±1 hours, with dissolved oxygen maintainedat 25% linked to agitation and aeration at 0.5 to 0.7 (V/V/M), with amixing speed of the fermentation broth of 80 to 160 RPM, preferably 100RPM, looped to dissolved oxygen at 25%. In-process controls to test forpurity and growth viability consisted of microscopic examination andstreaking of fermentation broth aliquots on GYM agar plates at 48, 72and 96 hour post-inoculation time points.

6.7. Harvesting and Extraction of the Fermentation Broth

Upon completion of the fermentation (FIG. 7), the pH of the fermentationbroth was adjusted in the pilot fermentor to a level of pH 3±0.1 by aslow addition of 99% H₂SO₄ with constant stirring. The fermentationbroth was then cooled in the pilot fermentor to 14±2° C. andsubsequently transferred into a holding tank and held in a refrigerationunit at 4±2° C. for a period of 16 to 72 hours. Upon completion of thecooling period, the cooled fermentation broth was then subjected toultrafiltration using a 0.2 to 0.45 micrometer pore size filter membrane(filtering time approximately 20 minutes) to produce a thick slurry ofharvested mycelia.

To establish values for a concentration and a mass of ECO-4601 in thefermentation broth on completion of the fermentation period, duplicate0.5 gram aliquots of the separated (filtered) mycelia were placed intoseparate 50 mL centrifuge tubes. Twenty mL of methanol were then addedto each tube, followed by sonification for 5 minutes and vortexing athigh speed for 30 seconds. Tubes were then centrifuged at 4000-5000 RPMfor 10-15 minutes, and the supernatant from each tube decanted into aclean 50 mL vial. The residual mycelia from each tube were re-extracted,as described above, using 20 mL of methanol. The two methanol extractsper tube were then combined (total volume approximately 40 mL), and a 1mL aliquot from the combined methanol extract was filtered through a0.45 μm filter membrane. A 10× dilution (in methanol) of the filteredaliquot was then prepared and submitted for quantitative analysis ofECO-4601 by HPLC-UV.

As a qualitative and quantitative check after completion of thefiltering of the acidified and cooled fermentation broth, the separatedbroth supernatant was also assayed for the presence of ECO-4601.Duplicate 5 mL sample aliquots of the broth supernatant were placed inseparate 50 mL centrifuge tubes followed by addition of 5 mL ethylacetate. Tubes were then vortexed at high speed for 1 minute, andcentrifuged at 4000-5000 RPM for 10-15 minutes. A 1 mL aliquot from theethyl acetate layer (upper phase) was withdrawn from each centrifugedtube, placed into a 10 mL glass culture tube and evaporated to drynessunder a stream of nitrogen. The dried residue in each tube wasre-dissolved in 1 mL of methanol and the resulting solution filtered.The filtrate from each tube was then subjected to HPLC-UV analysis forECO-4601 content.

ECO-4601 was extracted from the slurry of harvested mycelia by adding 3L of 100% methanol to every 1 L of the slurry, followed by circulationof the methanol-mycelia slurry mixture at a high velocity (from about 30Hertz to about 50 Hertz) through an ultrafiltration system for about60±20 min for an initial extraction round. The high circulating speedwas utilized to facilitate the breaking-up of mycelial aggregates, andthe temperature was allowed to increase to 42±3° C. during circulation.Optionally, an extended circulation time could be employed, for example120 minutes, provided however a lower circulation velocity is utilizedso as to avoid possible overheating and degradation of the extract. Oncethe mycelia slurry was properly mixed with the extraction solvent, thevalves of the ultrafiltration system were opened to allow the methanolextract to be collected. The methanol extract was fed into a containerand the residual mycelia re-extracted with a volume of methanol the sameas that used in the first extraction. Residual mycelia from the secondround of extraction were re-extracted with a volume of methanolapproximately three-eighths as that used per the first and secondextraction rounds. The circulating time for the third round ofextraction was reduced in comparison to the first two rounds ofextraction. The three methanolic extracts were pooled and evaporatedunder reduced pressure using a Büchi Rotavapor® Model R-153 (BÜCHILabortechnik AG, Flawil, Switzerland) to produce a thick, firstconcentrate.

Quantification of ECO-4601 contained in the methanolic extract per roundof extraction was performed as follows. Duplicate 1 mL aliquots of themethanol extract were taken, diluted by 10× with methanol and filtered.The filtered, diluted aliquots were then subjected to HPLC-UV analysisfor ECO-4601 content.

As a qualitative and quantitative check after completion of the methanolextraction of the mycelia, extracted waste mycelia (mycelia after thethird extraction round) were evaluated for ECO-4601 content. Duplicate 1gram samples of the extracted waste mycelia were placed into separate 50mL centrifuge tubes, followed by the addition of 10 mL of ethyl acetate.Tubes were then sonicated for 5 minutes, vortexed at high speed for 30seconds, and centrifuged at 4000-5000 RPM for 10-15 minutes. A 2 mLaliquot from the ethyl acetate layer (upper phase) was withdrawn fromeach centrifuged tube, placed into a 10 mL glass culture tube andevaporated to dryness under a stream of nitrogen. The dried residue ineach tube was re-dissolved in 1 mL of methanol and the resultingsolution filtered. The filtrate from each tube was then subjected toHPLC-UV analysis for ECO-4601 content.

Results from seven separate pilot fermentations are shown below in Table8. TABLE 8 Pilot Plant Fermentations Approximate Pilot- Amount ofconcentration Scale ECO-4601 in Mass of Mass of Mass of factor ECO-Fermentation Fermentation ECO-4601 ECO-4601 in ECO-4601 Mass of 4601from Batch Broth Upon in Extract Extract (2^(nd) in Extract ECO-4601Broth to 1^(st) No. Harvest (1^(st) Round) Round) (3^(rd) Round) inConcentrate Concentrate PE028 78.8 g 54.8 g 12.2 g 2.7 g 69.7 g 57 (175mg/L) PE029 76.5 g 43.3 g 12.7 g 4.4 g 60.4 g 51 (170 mg/L) PE031 67.1 g37.4 g 11.2 g 3.8 g 52.4 g 50 (149 mg/L) PE033 66.6 g 46.0 g  8.4 g 2.0g 56.4 g 54 (148 mg/L) PE034 57.2 g 33.6 g 11.3 g 1.8 g 46.7 g 52 (127mg/L) PE036 65.3 g 42.5 g 10.7 g 2.4 g 55.6 g 55 (145 mg/L) PE037 65.3 g42.5 g 16.0 g 3.1 g 61.6 g 61 (147 mg/L) PE038 76.1 g 49.3 g 16.3 g 3.9g 69.5 g 59 (169 mg/L)

The average volume of the first concentrate as shown in the last columnof Table 8, above, was about 7 L. Results from pilot plant fermentationsindicated that on a first round of extraction, the mass of ECO-4601contained in the methanol extract recovered from the fermentation broth,which could be evaporated to form a concentrate even after the initialround of extraction, ranged between about 55% to about 70% of the amountof ECO-4601 that was calculated to be present in the fermentation broth.On completion of all three rounds of extraction and with the combinationof the methanol extracts, the mass of ECO-4601 the present in the firstconcentrate ranged from about 78% to about 94% of the amount of ECO-4601that was calculated to be present in the fermentation broth.

Example 7 Increased ECO-4601 Production Using Different Culture Medium

7.1. General Procedure

A variety of culture medium were tested for their capability to providefor an elevated level of fermentation production of ECO-4601 relative toa particular preferred culture medium (HI). Small-scale fermentationcultures of Micromonospora sp. strain [S01U02]046 were grown under theconditions described above in Example 1 using the individual culturemedium as provided in Table 9, noted directly below. Ingredients foreach of the culture medium tested are noted in Table 1. For eachsmall-scale fermentation culture, harvesting and extraction of thefermentation broth also followed the procedure as provided in Example 1,as were the HPLC analyses of the extracted broth in order to quantifyproduction of ECO-4601 per each culture medium type used.

7.2. Results

The amount of ECO-4601 produced per culture medium type is expressed asan improvement factor relative to culture medium HI, the improvementfactor being calculated as a ratio of an average of the yield per media(mg/L) tested in two separate fermentation culture experiments (two 125mL flasks per media type per experiment) to the average of the yields intwo control cultures grown in HI culture medium. Results from thesesmall-scale fermentation culture experiments are summarized in Table 9.TABLE 9 Small-scale fermentation cultures using different culture mediumCulture medium JA DZ YB GP AP PI QP QI CA MI HI Improvement 4.45 4.3 3.53.3 3 3 2.7 2.7 2.7 2.3 / Factor (relative to HI media) Standard 2 0.41.3 N/A* 0 0.4 0.4 0.1 0.9 0.4 / Deviation (of Improvement Factor*Not Available - results are from one experiment.

Example 8 Scale Up Production-Large Volume/Pilot Plant Fermentation

Preparation of pilot manufacturing seeds stocks, working stock, workingseed plates, seed flasks, fermentation of inoculum fermentor, andfermentation in pilot fermentor were as described in Example 6.

8.1 Harvesting and Extraction of the Fermentation Broth

Upon completion of the fermentation, the pH of the fermentation brothwas adjusted in the pilot fermentor to a level of pH 3±0.1 by slowaddition of 99% H₂SO₄ with constant stirring. The fermentation broth wasthen cooled in the pilot fermentor to 4±2° C. and subsequentlytransferred into a holding tank and held in a refrigeration unit at 4±2°C. for a period of 16 to 72 hours. Upon completion of the coolingperiod, the cooled fermentation broth was subjected to either acontinuous centrifugation (5,000-20,000 RPM) or batch centrifugation(5,000-20,000 RPM) to produce a paste of mycelia. Calculations of theconcentration of the mass of ECO-4601 in the fermentation broth uponcompletion of the fermentation, and also upon post-collection of theslurry of harvested mycelia, were conducted as described in Example 6,and as also illustrated in FIG. 8. Optionally, upon completion of thecooling period, the cooled fermentation broth may be subjected toultrafiltration, as per the method described in Example 6, to produce aslurry of harvested mycelia.

8.2 Extraction of the Mycelia Paste

ECO-4601 was extracted from the paste of harvested mycelia with theaddition of 8 L (a range of 5 L to 10 L may be used) of 100% methanol toevery one kg of mycelial paste followed by mixing for approximately 30minutes±10 minutes at 250±50 RPM at 25±3° C., followed by circulation ofthe methanol-mycelia mixture in an filtration system as previouslydescribed in Example 6. Following a first round of extraction, theextracted mycelial paste was subjected to a second round of extractionof the residual mycelia using a volume of methanol as per the firstround of extraction. Quantification of ECO-4601 contained in themethanolic extract per round of extraction was performed as per Example6.

8.3 Displacement of Extracted ECO-4601 onto Adsorbent Resin

On completion of the rounds of extraction, the ECO-4601 methanolicextracts from each round were pooled, with mixing, into a stainlesssteel tank (compatible with organic solvents such as methanol) having avolume of approximately 2.5-3 times the volume of the pooled extract, asillustrated in FIG. 8. Pre-cleaned, dry, HP20 resin was then added tothe tank and mixed with the pooled extracts at a speed of 100-300 RPM atroom temperature (5° C.-40° C.). The amount of HP20 resin added wascalculated on the basis of 10 g of resin for every 1 g of ECO-4601calculated to be present in the pooled extract, although the amount ofresin that can be added can range from about 10 g to about 40 g forevery 1 g of ECO-4601 calculated to be present in the pooled extract.Upon completion of the second round of methanol extraction, theextracted mycelia (together with the internal surface of the containerand system in which the rounds of extraction were performed) were washedwith a further volume of 250 L of methanol to remove a residual amountof ECO-4601 that may have remained on the extracted mycelia or surface.The ECO-4601-bearing wash was thereafter combined with the first andsecond round extractions to form a pooled methanolic extract.

To displace the ECO-4601 from the pooled ECO-4601 methanolic extractsonto the adsorbent HP20 resin added to the pooled methanolic extract,water (USP-grade) was pumped into the tank (with mixing) at a volumetricrate of about 0.5% to about 20% of the volume of the pooled extracts perminute (i.e. for a 100 L pooled extract, water was pumped into the tankat a rate of abut 0.5 L to about 20 L per minute), and more preferablyat a rate of about 0.5% to about 5% of the volume of the pooled extractper minute. A total volume of water pumped into the tank was about 1.2to about 1.5 times the total volume of the pooled extract. To providesufficient displacement of the ECO-4601 from the pooled methanolicextracts onto the HP20 resin, the total volume of water to be pumpedinto the tank may comprise a volume that is about equal to the totalvolume of the pooled extract. Upon completion of the pumping the totalvolume of water into the tank containing the pooled ECO-4601 methanolicextracts/HP20 resin mixture, the pooled extract/HP20 resin/water mixturewas allowed to incubate at room temperature (5° C.-40° C.) with rotation(100 RPM to 300 RPM) for a period of about 7.5 minutes. Optionally, themixture may be allowed to incubate for a period of about 5 minutes toabout 60 minutes. Upon completion of the incubation period, the HP20resin bearing the adsorbed ECO-4601 was thereafter recovered byfiltration, for example, by using a Hayward bag filter assembly unit;type FBF-0102-AB10 (Hayward, Inc.) equipped with a 50 μm Hayward bagfilter membrane (GAF nylon filter, Part #: PO2Z-E-20S), as would bepracticed by a person of skill in the art. On completion of the recoveryof the ECO-4601 from the resin, the amount of ECO-4601 remaining in thepooled methanol extracts was assayed for by taking duplicate 1 mLaliquots of the methanol extract, followed by HPLC-UV analysis up thefiltered diluted aliquots for ECO-4601 content.

8.4 Results

Results from a pilot fermentation wherein ECO-4601 was extracted anddisplaced onto an adsorbent resin using the water displacement techniqueas described above are shown below in Table 10. TABLE 10 Pilot PlantFermentation (1000 L) Amount of ECO-4601 in Mass of Mass Mass ofFermentation ECO-4601 of ECO-4601 Mass of ECO-4601 Mass of Broth Upon inExtract in Extract ECO-4601 in Waste ECO-4601 in Harvest (1^(st) Round)(2^(nd) Round) from wash Mycelia Concentrate 125.1 g 85.3 g 11.4 g 25.2g 3.2 g 121.9 g

Results from the pilot plant fermentation indicated the mass of ECO-4601recovered from the fermentation broth, as calculated to be present onthe extract-bound resin recovered from the pooled methanol extracts uponcompletion of the displacement step, to be approximately 97.4% of theamount of ECO-4601 that was calculated to be present in the fermentationbroth.

Example 9 Downstream Processing of Resin-Adsorbed FarnesylatedDibenzodiazepinone

A first concentrate comprising the resin-absorbed farnesylateddibenzodiazepinone can be subjected a further downstream processing inorder to reduce a level of an impurity, such an impurity potentiallyhaving a deleterious effect or effects such as upon the crystallizationof the ECO-4601. To reduce the level of such an impurity, ECO-4601 thathas been displaced onto the adsorbent resin was subjected to rounds ofcolumn cleanup, wherein for a first-round cleanup, an HP20 column wasprepared by transferring 2.5±0.2 kg of pre-cleaned HP20 in 20% aqueousmethanol to a 30 L capacity column and the column thereafter eluteduntil the HP20 resin was settled in the column so as to form a resin bedin the column. HP20 resin bearing approximately 315 grams of ECO-4601(colour of the HP20 resin bearing the ECO-4601 was a reddish tobrownish/dark brown) was then transferred into the column withoutdisturbing the clean bed of HP20. The HP20 resin bearing theapproximately 315 grams of ECO-4601 resulting from the displacementtreatment, as described above in Example 8, of extract having 339.2grams of ECO-4601. Upon completion of the transfer of the HP20 resinbearing the 315 grams of ECO-4601 into the column, the column was theneluted with 150±5 L of 65% aqueous methanol at a flow rate of 400 mL/minand the effluent from the elution with the 65% aqueous methanol wasdiscarded. After the 65% aqueous methanol elution step, the column wasthen eluted with 150±5 L of 70% aqueous methanol at a flow rate of 400mL/min and the effluent collected in 20 L fractions. Upon completion ofthe 70% aqueous elution, the column was thereafter eluted with 100±5 Lof 90% aqueous methanol at a flow rate of 400 mL/min and the effluentcollected in 20 L fractions. The 20 L fractions from the 70% aqueousmethanol and the 90% aqueous methanol elution steps were assayed for thepresence of ECO-4601, and fractions having greater than or equal to 1%ECO-4601 were combined so as to form a pool of aqueous methanolfractions containing ECO-4601 from the first-round cleanup. The pool ofaqueous methanol fractions containing approximately 304 grams ofECO-4601 was then subjected to a second-round cleanup.

For the second-round cleanup, the pool of aqueous methanol fractionscontaining the approximately 304 grams of ECO-4601 from the first-roundcleanup was transferred into a 350 L mixing tank. Pre-cleaned HP20 resinwas then added to the mixing tank in an amount of 18±2 L with rotationof the mixer at 120±10 RPM. Water (UPS-grade) was thereafter pumped intothe tank at a volumetric rate of 2±0.5 L/min to a total volume equal tothe volume of the pooled methanol fractions from the first-roundcleanup. On completion of the pumping of the water into the tank, theHP20 bearing the ECO-4601 was recovered using a bag filter assembly unitand filter as described in Example 8. For the second-round cleanup, anHP20 column was prepared by transferring 2.5±0.2 kg of pre-cleaned HP20in 5 L of 20% aqueous methanol to a column, after which the columnwashed with 20% aqueous methanol and the HP20 resin allowed to settle inthe column so as to form a resin bed in the column. After settling ofthe column was completed, the HP20 resin onto which the ECO-4601 hadbeen displaced was transferred to the column and the column washed with30% aqueous methanol. Thereafter, the column was eluted with 150±5 L of65% aqueous methanol at a flow rate of 400 mL/min and the effluent fromthe elution with the 65% aqueous methanol was discarded. After the 65%aqueous methanol elution step, the column was then eluted with 150±5 Lof 70% aqueous methanol at a flow rate of 400 mL/min and the effluentcollected in 20 L fractions. Upon completion of the 70% aqueous methanolelution, the column was thereafter eluted with 100±5 L of 90% aqueousmethanol at a flow rate of 400 mL/min and the effluent collected in 20 Lfractions. The 20 L fractions from the 70% and the 90% aqueous methanolelution steps were assayed for the presence of ECO-4601, and fractionshaving greater than or equal to 1% ECO-4601 were combined so as to forma pool of aqueous methanol fractions containing ECO-4601 from thesecond-round cleanup. The pool of aqueous methanol fractions containingapproximately 278 grams of ECO-4601, and the pool of aqueous methanolfractions was thereafter evaporated to provide a second concentratehaving a grayish to grayish-brown colour. While the level of anyparticular impurity that may remain in the second concentrate after theprocessing may vary, the second concentrate is to be considered to besubstantially free of molecules other than the farnesylateddibenzodiazepinone of Formula I. From the second concentrate,acrystalline farnesylated dibenzodiazepinone of Formula I may beproduced upon subjecting the second concentrate to a crystallizationprocess.

Example 10 Crystallization

A second concentrate may be utilized for the production of crystallineECO-4601. A 33% ethanol crystallization solution can be prepared in a500 L crystallization tank, with stirring at 140±20 RPM. The temperatureof the crystallization solution is allowed to equilibrate at 30±2° C.for 120±10 minutes. A sample solution for crystallization can beprepared by dissolving the second concentrate (resulting from the tworounds of cleanup processing the HP20 column (as described above inExample 8)) by mixing the second concentrate in absolute ethanol (24±3g/L). The second concentrate is to be mixed well with the absoluteethanol in order to dissolve the second concentrate into the absoluteethanol, for example, by manual mixing for 7.5±2.5 minutes. Purifiedwater is then added to the ethanolic solution while stirring and theresulting solution filtered through a 0.45 μm to 10 μm membrane filter.The sample solution may then be pumped into the crystallization tank ata rate (mL/min) that is about 10 mg/min/10 L of crystallization solutionvolume. During the pumping of the crystallization solution, nitrogen gasor purified air is to be flowed over the crystallization tank at a flowrate of 20±2 L/min. The temperature of the tank is to be maintained at30±2° C. while mixing the sample solution with the 33% ethanolicsolution at 40±10 RPM. As the sample solution is being pumped into thecrystallization tank, the crystallization tank is to be seeded with75±25 mg of ECO-4601 in water, and the delivery of the sample solutioninto the crystallization tank completed. ECO-4601 crystals are to beallowed to mature for 14±2 hours, with nitrogen gas flowing over thecrystallization tank at a flow rate of 10±2 L/min and mixing occurringat 40±10 RPM while the tank temperature is maintained at 30±2° C.Purified water is to thereafter be pumped into the crystallization tankat a rate (mL/min) about equal to the volume of water to be addeddivided by 1200, and the crystals allowed to mature for 24±4 hours withnitrogen gas flowing over the crystallization tank at a flow rate of10±2 L/min and mixing occurring at 40±10 RPM while the tank temperatureis maintained at 30±2° C. At the completion of the maturity period, themixing is to be stopped and crystals allowed to settle for 18±6 hours.To harvest the settled crystals from the crystallization tank, thesupernatant from the crystallization tank is to be drained into aholding tank and the settled crystals (in the form of a slurry) are tobe drained into a receiving tank and recovered by filtration through a10-20 μm sintered glass filter funnel. Recovered crystals are then to bere-suspended in cold (about 4° C.) 10% ethanol and filtered again, withthe resuspension and filtering being twice repeated. The crystals arethen to be allowed to anneal in a vacuum oven (operating at about atleast equal to or greater than a pressure of 25 inches of Hg, 68±2° C.)for 21±5 hours. On completion of the annealing, the annealed crystals(having a needle-like shape) may then be transferred to a container,preferably a polypropylene container, and stored at 25±2° C.

Example 11 Demonstration of a Purification of a Specific Batch Resultingin an Incremental Purity Level of ECO-4601 Relative to the Purity of theECO-4601 in the Fermentation Broth

As an example of a measure of an increase in a purity level of ECO-4601from a fermentation broth stage to production of crystalline ECO-4601, apilot scale fermentation broth batch (record number DR00116) (producedin accordance with the procedure described in Example 6) of 450 L(having a mss of about 450 kg) was calculated to contain about 78 gramsof ECO-4601. A cell mass of the microorganism producing the farnesylateddibenzodiazepinone of Formula I of about 22.5 kg (comprising about 5% ofthe mass of the fermentation broth upon completion of the fermentationprocess) was calculated to be present in the broth. As such, uponcompletion of the fermentation period, a purity level of about 0.35% ofECO-4601 was calculated to be present in the fermentation broth (78grams divided by (0.05×450,000 grams). Levels of purity of the ECO-4601in the fermentation broth upon completion of the fermentation period maybe estimated to range from at least about 0.3% to about 1%. Uponsubjecting the first concentrate (generated from treatment of theextract of the fermentation broth) to a first round of column cleanup,the calculated purity of the ECO-4601 resulting from such first round ofcleanup was about 66%, and upon completion of the second round of columncleanup (as described above in Example 9) the purity of the ECO-4601comprising the so-formed second concentrate was calculated to be about88.5%. Thereafter, upon subjecting the second concentrate to thecrystallization process, the resultant crystals were calculated to beabout 99% ECO-4601.

In case of conflict, the present specification, including definitions,will control. While this invention has been particularly shown anddescribed with reference to the preferred embodiments thereof, it willbe understood by those skilled in the art that various changes in formand details may be made without departing from the scope of theinvention encompassed by the appended claims.

All documents, publications and other materials cited herein, includingbut not limited to, patents, patent application publications, journalarticles, books, product literature and manuals, are expresslyincorporated herein by reference in their entirety.

1. A process for recovering and concentrating a farnesylateddibenzodiazepinone of structural Formula I

comprising: a) fermenting a strain of a microorganism capable ofproducing a farnesylated dibenzodiazepinone of structural Formula I inan aqueous culture medium, thereby producing a fermentation brothcomprising said farnesylated dibenzodiazepinone; b) adjusting saidfermentation broth to allow said farnesylated dibenzodiazepinone toassociate with a particulate matter present in said broth; c) harvestingsaid particulate matter from said broth, thereby obtaining a harvestedparticulate matter; d) extracting said harvested particulate matter witha volume of a suitable organic solvent in a proportion of about 2:1 toabout 5:1 to said harvested particulate matter, thereby forming anextract; and e) treating said extract to form a first concentratecomprising said farnesylated dibenzodiazepinone of Formula I, whereinsaid first concentrate comprises said farnesylated dibenzodiazepinone ata concentration greater than about 50-fold than said farnesylateddibenzodiazepinone in said fermentation broth of a), and wherein saidfarnesylated dibenzodiazepinone is recovered from said fermentationbroth of a) in an amount of at least about 50% of the amount of saidfarnesylated dibenzodiazepinone in said fermentation broth.
 2. Theprocess of claim 1, wherein said farnesylated dibenzodiazepinone isrecovered from said fermentation broth of a) in an amount of at leastabout 60% of the amount of said farnesylated dibenzodiazepinone in saidfermentation broth.
 3. The process of claim 1, wherein said farnesylateddibenzodiazepinone is recovered from said fermentation broth of a) in anamount of at least about 65% of the amount of said farnesylateddibenzodiazepinone in said fermentation broth.
 4. The process of claim1, wherein the particulate matter comprises the microorganism present inthe fermentation broth.
 5. The process of claim 4, wherein theparticulate matter further comprises an adsorbent resin A.
 6. Theprocess of claim 4 or 5, wherein the fermentation broth is adjusted inb) to a pH value of about 2 to about
 4. 7. The process of claim 6,wherein the broth is further adjusted to a temperature range of about 2°C. to about 10° C.
 8. The process of claim 7, wherein the broth ismaintained at the temperature range for a period of about 16 hours toabout 72 hours.
 9. The process of claim 1, wherein the fermentation iscompleted in from about 48 hours to about 110 hours.
 10. The process ofclaim 1, wherein the microorganism is a bacterium.
 11. The process ofclaim 10, wherein the bacterium is an Actinomycete.
 12. The process ofclaim 11, wherein the Actinomycete is a Micromonospora, a Streptomycesor a Rhodococcus species.
 13. The process of claim 12, wherein theActinomycete is Micromonospora sp. [S01]046 having IDAC accession number231203-01 or Micromonospora sp. [S01U02]046 having IDAC accession number070905-01.
 14. The process of claim 1, wherein the suitable organicsolvent is selected from the group consisting of i) at least one loweralkyl alcohol; ii) ethyl acetate; and iii) acetonitrile.
 15. The processof claim 14, wherein the lower alkyl alcohol is selected from the groupconsisting of ethanol, propanol, iso-propanol, butanol or methanol, anda mixture thereof.
 16. The process of claim 15, wherein the lower alkylalcohol is methanol.
 17. The process of claim 1, wherein the aqueousculture medium is AP, CA, DZ, GP, HI, JA, MI, PI, QI, QP or YB, as shownin Table
 1. 18. The process of claim 1, wherein the aqueous culturemedium is QB, MA, KH, RM, JA, FA, or HI, as shown in Table 1 and Table2.
 19. The process of claim 1, wherein the harvesting of (c) isperformed using an ultrafiltration system.
 20. The process of claim 19,wherein the ultrafiltration system has one or more filter elementshaving a pore size of about 0.2 to about 0.45 micrometers.
 21. Theprocess of claim 1, wherein the harvesting of (c) is performed bycentrifugation.
 22. The process of claim 1, wherein the extracting of(d) comprises one to three rounds of extraction using the suitableorganic solvent.
 23. The process of claim 22, wherein the extractingfurther comprises circulating the particulate matter together with thesuitable organic solvent in a system at a flow rate of about 30 Hz toabout 50 Hz, and at a temperature of about 39° C. to about 45° C. 24.The process of claim 23, wherein the circulation is for a period ofabout 50 minutes to about 120 minutes.
 25. The process of claim 22,wherein the extraction comprises subjecting the particulate mattertogether with the suitable organic solvent to a centrifugationtreatment.
 26. The process of claim 1, wherein the volume of thesuitable organic solvent is calculated in proportion to the harvestedparticulate matter mass (volume:mass).
 27. The process of claim 1,wherein the volume of the suitable organic solvent is calculated inproportion to the harvested particulate matter volume (volume:volume).28. The process of claims 1, wherein the treating of (e) is anevaporation treatment.
 29. The process of claim 28, wherein theevaporation treatment is conducted under a reduced pressure.
 30. Theprocess of claim 28, wherein the evaporation treatment is performedwhile incubating the extract with an adsorbent resin B.
 31. The processof claim 30, wherein the evaporation treatment is conducted under areduced pressure.
 32. The process of claim 30 or 31, wherein theadsorbent resin B is HP20.
 33. The process of claim 1, wherein thetreating of (e) comprises incubating the extract with an absorbent resinC to form a mixture, followed by an addition of water to the mixture todisplace the farnesylated dibenzodiazepinone of Formula I onto the resinC.
 34. The process of claim 33, wherein the adsorbent resin C mixed withthe extract is provided in a ratio (W/W) of about 10 times to about 40times to the farnesylated dibenzodiazepinone of Formula I in theextract.
 35. The process of claim 33 or 34, wherein the water is addedto the mixture at a volume flow rate (V/V) of about 0:5% to about 20% ofthe volume of the mixture.
 36. The process of claim 35, wherein thevolume flow rate is about 0.5% to about 5% of the volume of the mixture.37. The process of claim 35, wherein the water added to the mixture isof a total volume of about 1.0 to about 1.5 times the volume of themixture.
 38. The process of claim 37, wherein the total volume of wateradded to the mixture is of about 1.2 to about 1.5 times the volume ofthe mixture.
 39. The process of claim 1, wherein said fermentation brothof a) comprises about 10 mg/L to about 465 mg/L of said farnesylateddibenzodiazepinone.
 40. The process of claim 1, further comprisingprocessing said first concentrate obtained by the method of claim 1 toreduce a level of an impurity in said first concentrate to therebyproduce a second concentrate.
 41. The process of claim 40, furthercomprising the step of crystallizing from said second concentrate thefarnesylated dibenzodiazepinone of Formula I to thereby produce acrystal of the farnesylated dibenzodiazepinone of Formula I suitable foruse in the preparation of a pharmaceutical formulation.
 42. Aconcentrate obtained by the method of claim
 1. 43. The concentrate ofclaim 42, wherein said farnesylated dibenzodiazepinone is recovered fromsaid fermentation broth of a) in an amount of at least about 60% of theamount of said farnesylated dibenzodiazepinone in said fermentationbroth.
 44. The concentrate of claim 42, wherein said farnesylateddibenzodiazepinone is recovered from said fermentation broth of a) in anamount of at least about 65% of the amount of said farnesylateddibenzodiazepinone in said fermentation broth.
 45. The concentrate ofclaim 42, wherein said fermentation broth comprises a microorganismcapable of producing said farnesylated dibenzodiazepinone.
 46. Theconcentrate of claim 45, wherein said microorganism is a bacterium. 47.The concentrate of claim 46, wherein said bacterium is an Actinomycete.48. The concentrate of 47, wherein said Actinomycete is aMicromonospora, a Streptomyces or a Rhodococcus species.
 49. Theconcentrate of claim 48, wherein the Actinomycete is Micromonospora sp.[S01]046 having IDAC accession number 231203-01 or Micromonospora sp.[S01U02]046 having IDAC accession number 070905-01.
 50. The concentrateof claim 42, wherein the extract is mixed with an adsorbent resin D toform a mixture, wherein the resin D is added to the extract in a ratio(W/W) of about 10 times to about 40 times of the farnesylateddibenzodiazepinone of Formula I present in said extract, and whereinwater is added to the mixture at a volumetric rate (V/V) of about 0.5%to about 20% of the volume of the mixture.
 51. A microorganism capableof producing, during a fermentation period in a volume of an aqueousculture medium, a farnesylated dibenzodiazepinone of structural FormulaI

at a yield rate of about 0.073 mg/Uh to about 5.06 mg/Uh as averagedover the fermentation period.
 52. The microorganism of claim 51, whereinthe yield rate is of about 2.15 mg/Uh to about 5.06 mg/Uh as averagedover the fermentation period.
 53. The microorganism of claim 51, whereinthe yield rate is of about 3.00 mg/Uh to about 5.06 mg/L/h as averagedover the fermentation period.
 54. The microorganism of claim 51, whereinsaid microorganism is a bacterium.
 55. The microorganism of claim 54,wherein said bacterium is an Actinomycete.
 56. The microorganism of 55,wherein said Actinomycete is a Micromonospora, a Streptomyces or aRhodococcus species.
 57. The microorganism of claim 56, wherein theActinomycete is Micromonospora sp. [S01]046 having IDAC accession number231203-01 or Micromonospora sp. [S01U02]046 having IDAC accession number070905-01.
 58. The microorganism Micromonospora sp. [S01U02]046 havingIDAC accession number 070905-01.